Name: protocol for the direct conversion of murine embryonic fibroblasts into trophoblast stem cells
Uploaded: 2016-07-25T15:00:00.000+00:00
Duration: 8 min 57 s
Description: Watch this Scientific Journal Video about Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells at JoVE.com

<p class="jove_content"> Tutti gli esperimenti di topo sono stati condotti secondo la legge tedesca di protezione degli animali e in accordo con l&#39;approvazione dei comitati per la cura degli animali istituzionali locali (Landesamt fuer Natur, Umwelt und Verbraucherschutz, Nord Reno-Westfalia [numero ID di approvazione: AZ 84-02.04.2013 .A428]). </p><p class="jove_title"> 1. Carta Preparazione </p><ol><li> Preparare 293T media: Modified Eagle Medium di Dulbecco (DMEM), 10% (v / v) di FBS, L-glutamina (2 mM), piruvato di sodio (1 mM), penicillina / streptomicina (1x). </li><li> Preparare medio MEF: DMEM, 10% (v / v) di FBS, L-glutamina (2 mM), aminoacidi non essenziali 1x, penicillina / streptomicina (1x). </li><li> Preparare medio TS (w / o FGF4 / eparina): Roswell Park Memorial Institute media (RPMI) 1640, il 20% (v / v) di FBS (cellule staminali cultura grado), penicillina / streptomicina (1x), L-glutammina (2 mm ), piruvato di sodio (1 mM), 2-mercaptoetanolo (0,1 mM). </li><li> Preparare medio TS con FGF4 / eparina (TS + F4H): RPMI 1640, il 20% (v / v) di FBS (cellule staminalicultura grade), penicillina / streptomicina (1x), L-glutamina (2 mM), piruvato di sodio (1 mM), 2-mercaptoetanolo (0,1 mM), 25 ng / ml FGF4, 1 mg / ml di eparina. <br /> Nota: FGF4 ed eparina sono aggiunti direttamente nelle supporto immediatamente prima dell&#39;uso. </li><li> Preparare TS-CM con FGF4 / eparina (TS-CM + F4H): 70%-MEF condizionata TS media + 30% preparata al momento TS media + 25 ng / ml FGF4, 1 mg / ml di eparina. <br /> Nota: FGF4 ed eparina sono aggiunti direttamente nelle supporto immediatamente prima dell&#39;uso. </li><li> Preparare medio Trasfezione: Avanzato DMEM, 2% (v / v) di FBS (radice di grado coltura cellulare), L-glutammina (2 mm), la penicillina / streptomicina (1x). </li><li> Preparare medio Virus-produzione: Avanzato DMEM, 5% (v / v) di FBS (radice di grado coltura cellulare), L-glutammina (2 mm), la penicillina / streptomicina (1x). </li></ol><p class="jove_title"> 2. murino embrionale Fibroblasto Derivazione </p><ol><li> Set accoppiamenti a tempo utilizzando peso 129S2SV femmine e maschi omozigoti topi R26 :: rtTA (nome ceppo; B6.Cg-Gt (Rosa) 26Sor <sup>TM1 (rtTA</sup>* M2) Jae / J 7). Su E13.5 topi sacrificio dislocazione cervicale e isolare fibroblasti primari murini embrionali (MEF) secondo protocolli standard <sup>8.</sup> Successivamente, le cellule piastra da un embrione su un piatto di 150 millimetri in MEF media. </li><li> Una volta che le culture MEF primarie raggiungano la confluenza su piatti di 150 mm, lavare le cellule due volte con 10 ml di PBS e aggiungere 4 ml 0,05% soluzione di tripsina / EDTA. Incubare piatti per 3 - 4 minuti in incubatrice a 37 ° C. </li><li> Dopo incubazione verifica se le cellule staccate dal piatto utilizzando un microscopio invertito (utilizzando un obiettivo 10X). Aggiungere 5 ml di media MEF per fermare tripsinizzazione e raccogliere sospensione cellulare in un tubo conico da 50 ml. </li><li> sospensione cellulare Pool da diversi piatti raccogli in una provetta conica da 50 ml. sospensione cellulare centrifugare per 3 minuti a 200 xg e 10 ° C. </li><li> Gettare il surnatante e risospendere pellet in cella FBS contenente il 10% (v / v) dimetilsolfossido. Congelare MEF in aliquote per un ulteriore uso. <br /> Nota: In alternativa, utilizzare tip selvaggioe MEF primarie; tuttavia, un ulteriore vettore lentivirale che esprime rtTA deve essere trasdotte insieme agli altri vettori lentivirali (per esempio usando il plasmide FUdeltaGW-rtTA descritto da Maherali <em>et al.</em> <sup>9).</sup> </li><li> <strong>Preparazione del mezzo TS MEF condizionata</strong> <br /> Nota: Prima di esperimenti di conversione, MEF condizionata (CM) TS supporto deve essere preparato, che è richiesto per sottocultura di singole linee ITSC (descritti nella sezione 6). <ol><li> Piastra 1 x 10 <sup>7</sup> MEF mitoticamente inattivati ​​al piatto 150 mm di media MEF sul numero desiderato di piatti. Inattivare MEF di γ-irraggiamento con una dose di 9 Gy. <br /> Nota: La resa per piatto 150 millimetri è di circa 60 ml di CM. </li><li> Il giorno dopo la placcatura, aspirare e scartare medio MEF e aggiungere 20 ml preparati al momento medie TS (senza FGF4 / eparina) su ogni piatto di MEF e luogo piatti mitoticamente inattivato di nuovo nel incubatore a 37 ° C. </li><li> dopo l&#39;incubazionemedia TS per tre giorni sul MEF mitoticamente inattivati, raccogliere media in 50 ml provette coniche. Filtrare media attraverso un filtro 0,2 micron e procedere con la 2.6.4. Sostituire CM con 20 ml di terreno fresco TS per la preparazione di un ulteriore lotto di CM. <br /> Nota: mitoticamente MEF inattivati ​​possono essere utilizzati per la preparazione CM fino a tre volte. </li><li> Aliquota 35 ml di CM filtrato in 50 ml provette coniche e conservare a -20 ° C fino al momento dell&#39;uso. Dopo lo scongelamento, aggiungere 15 ml di terreno TS preparati al momento al fine di ottenere il 70% TS-CM e conservare a 4 ° C per un massimo di un mese. </li></ol></li></ol><p class="jove_title"> 3. vettori lentivirali Produzione in cellule 293T </p><p class="jove_content"> Nota: Tutti i passaggi vengono eseguiti nello spazio di laboratorio in licenza a livello di biosicurezza 2. </p><ol><li> Prima di esperimenti di conversione, preparare vettori lentivirali per la doxiciclina (DOX) dipendenti over-espressione di Tfap2c, GATA3, Eomes, ETS2 e mCherry. Per ogni lentivirus, le cellule 293T co-trasfezione utilizzando ilrispettivo vettore di trasferimento lentivirale insieme ad un plasmide imballaggio e una busta esprimendo plasmide VSV-G (per informazioni plasmide riferimento alla Tabella Materials). <br /> Nota: per informazioni utili per quanto riguarda la produzione di virus si riferiscono a Gavrilescu <em>et al <sup>10..</sup></em> Anche se descrive la produzione di retrovirus, osservazioni di carattere generale in materia di qualità delle cellule 293T e il DNA plasmide sono vere per la produzione di lentivirus pure. <br /> ATTENZIONE: le misure di sicurezza appropriate devono essere prese, quando si lavora con vettori lentivirali e il lavoro deve essere eseguito in una struttura biosicurezza di livello 2. </li><li> Coat cinque 100 piatti mm con soluzione coprendo i piatti con 5 ml di soluzione e delicatamente dondolo per distribuire uniformemente soluzione di rivestimento di poli-L-lisina (100 mg / ml). Porre le capsule di nuovo nel incubatore a 37 ° C per 30 min. Dopo 30 min soluzione di rivestimento aspirare e lavare i piatti una volta con PBS. </li><li> Tavola 7 x 10 <sup>6</sup> 293T cellule per piatto a 10 ml mezzi 293T-cellule, spiatti Wirl per distribuire uniformemente le cellule e posto i piatti di nuovo nel incubatore a 37 ° C. </li><li> La mattina seguente, sostituire terreno di coltura con 5 ml di terreno trasfezione. Aggiungere 6 ml di una 1,000x magazzino di soluzione clorochina (25 mm) e posizionare i piatti di nuovo nel incubatore a 37 ° C. </li><li> <strong>Nel frattempo, preparare la miscela di trasfezione:</strong> <ol><li> Per ogni vettore lentivirale preparare due 5 ml provette, etichettate A e B. </li><li> Aggiungere 600 ml di 2x HEPES Buffered Saline (HBS) per tubo A. </li><li> Nel mix tubo B 61,5 ml soluzione di CaCl <sub>2</sub> (2 m), con 18,5 mcg lentivector DNA e 9,25 mg psPAX2 e 9,25 mg di pMD2.G. Regolare il volume a 600 ml con la cultura sterile di grado H <sub>2</sub> O. </li><li> Aggiungere la soluzione dal tubo di B goccia a goccia a tubo A mentre vortex. Incubare miscela di trasfezione per 15 minuti a RT. </li></ol></li><li> Aggiungere miscela di trasfezione con molta attenzione cadere saggio le cellule 293T preparati sul piatto a 100 mm. UNopo 5 a 6 ore rimuovere medie e sostituire con 10 ml di terreno virus-produzione. Mettere i piatti di nuovo nel incubatore a 37 ° C. <br /> Nota: Fare attenzione durante la rimozione e la sostituzione di media da cellule 293T facilmente si staccano dal piatto. variazione media 5-6 ore dopo la trasfezione è fondamentale, dal momento che l&#39;esposizione prolungata alla clorochina è tossico per le cellule. </li><li> La mattina seguente, rimuovere con attenzione e gettare media e sostituirlo con 7 ml di terreno virus-produzione. </li><li> Il mattino successivo alla vendemmia il primo lotto di media virus contenenti. Aspirare media e raccogliere in un tubo da 15 ml. Ancora una volta, aggiungere 7 ml di mezzo di virus-produzione sulle celle. Conservare virus contenente il mezzo in frigo fino a quando il secondo lotto è raccolto il giorno seguente. </li><li> La mattina successiva aspirare il secondo lotto di terreno contenente il virus aggiungendola ai 15 ml tubo conico contenente il primo lotto. cellule 293T possono ora essere scartati. Filtro virus contenente il mezzo attraverso un sur 0,45 micronacetato di cellulosa senza factant filtro -membrane. Aliquota filtrato, virus contenenti medio e conservare a -80 ° C per un ulteriore uso. <br /> Nota: la produzione di virus può essere eseguita anche in piatti 6 pozzetti. Ridimensionare volumi e numero di cellule di conseguenza. </li></ol><p class="jove_title"> 4. fibroblasti trasduzione con 4 fattori e mCherry controllo vettoriale </p><p class="jove_content"> Nota: Tutti i passaggi vengono eseguiti in una struttura di biosicurezza di livello 2. </p><ol><li> Per una rappresentazione schematica del contorno sperimentale riferimento alla <strong>Figura 1.</strong> Scongelare un&#39;aliquota di primarie rtTA-MEF e piastra 3,33 x 10 <sup>5</sup> cellule per pozzetto di un piatto 6 pozzetti in un volume totale di 2 ml di supporti MEF. Preparare almeno 3 pozzi ed etichettarli con 4 fattori (4F), mCherry e controllo. <br /> Nota: In alternativa, MEF tipo selvatico possono essere utilizzati, quando un rtTA esprime vettori lentivirali viene aggiunto al cocktail lentivector durante la fase 4.2. </li><li> Il giorno seguente dopo che le cellule pienamente recuperato da cryopreconservazione, sostituire i media MEF nelle tre 6-pozzi con 0,6 ml (4F), 0,9 ml (mCherry) e 1 ml (controllo) dei mezzi di trasduzione, rispettivamente. Scongelare una aliquota di PLV-teto-Tfap2c, -Gata3, -Eomes, ets e virus -mCherry a temperatura ambiente e aggiungere 100 ml di ogni surnatante virus contenenti (Tfap2c, GATA3, Eomes, ETS2) nel 4F bene. <br /> Nota: In caso di MEF wild type, ridurre il volume dei mezzi di comunicazione preplated da 100 ml e inoltre aggiungere 100 ml di virus FUdeltaGW-rtTA contenente surnatante al mix 4F. </li><li> Aggiungere 100 ml di mCherry supporti virus contenenti al pozzo etichettato con mCherry. Aggiungere polibrene ad una concentrazione finale di 8 mg / ml in ogni pozzetto. Porre la capsula posteriore in incubatore a 37 ° C e incubare MEF O / N con surnatante contenente il virus. <br /> Nota: In caso di MEF wild type, ridurre il volume dei mezzi di comunicazione preplated da 100 ml e inoltre aggiungere 100 ml di FUdeltaGW-rtTA surnatante virus contenenti. </li><li> La mattina seguente, rimuovere con attenzionemedia e contenente il virus, lavare tre volte con 2 ml di PBS. Infine, aggiungere 2 ml di TS Media (senza FGF4 / eparina). <br /> Nota: MEF trasdotte possono sia essere utilizzati direttamente per esperimenti di induzione ITSC o congelati per un ulteriore uso. </li></ol><p class="jove_title"> 5. fibroblasti di conversione ITSC </p><p class="jove_content"> Nota: Tutti i passaggi vengono eseguiti nello spazio di laboratorio in licenza a livello di biosicurezza 2. </p><ol><li> Lavare MEF trasdotte due volte con 2 ml di PBS e dopo l&#39;aggiunta soluzione di 0,5 ml di tripsina / EDTA incubare in incubatrice per 3 - 4 minuti. Controllare sotto un microscopio invertito per un corretto distacco delle cellule. <ol><li> Inattivare tripsina con l&#39;aggiunta di 1 ml di TS media. Trasferire la sospensione cellulare in un tubo da 15 ml e centrifugare per 3 min a 200 x g. Eliminare il surnatante e risospendere le cellule in 2 ml di TS media. Utilizzare il campione di controllo non trasdotte per contare le cellule. </li><li> Piastra 4F-MEF, mCherry-MEF e MEF di controllo non trasdotte ciascuno ad una densità di 2 x 10 <sup>5</sup>cellule per ogni 100 mm piatto di coltura cellulare in 10 ml TS mezzo contenente 2 mg / ml DOX (media TS + FGF4 / eparina + dox). </li><li> In alternativa, eseguire transdifferenziazione su piatti più piccoli. Tavola 3.6 x 10 <sup>3</sup> cellule per cm <sup>2</sup> sulle dimensioni piatto desiderato, il volume scala di conseguenza. </li></ol></li><li> Il giorno successivo, usando un microscopio a epifluorescenza (utilizzando un obiettivo 10X e un filtro per fluorescenza rossa eccitazione) monitorare l&#39;espressione di proteine ​​mCherry per valutare l&#39;efficienza di trasduzione. <br /> Nota: l&#39;efficienza di trasduzione dovrebbe essere prossimo al 100%. Se aumento della morte cellulare è evidente dopo l&#39;induzione del transgene questo significa che il virus titolo era troppo alto. Trasduzione deve essere ripetuto con ridotta quantità di mezzo contenente il virus. </li><li> D&#39;ora in poi il cambiamento media su tutti i piatti a giorni alterni fino al giorno 10 dopo la placcatura. Successivamente, nutrire le cellule con mezzo di TS senza DOX (media TS + FGF4 / eparina). </li><li> L&#39;utilizzo di un monitor microscopio invertito percomparsa di &#39;transdifferentiated zone&#39; (fare riferimento alla <strong>figura 2B e C)</strong> sul piatto 4F per un massimo di 3 settimane. Tali colonie non dovrebbero essere visibili sui due piatti di controllo (mCherry-MEF ei MEF non trasdotte). </li></ol><p class="jove_title"> 6. L&#39;isolamento di singoli ITSC Lines </p><p class="jove_content"> Nota: Per il passaggio 6.3 non è richiesta una cappa coltura di tessuti. Si raccomanda di pulire il microscopio invertito con il 70% di etanolo per ridurre al minimo le possibilità di contaminazione batterica. colonie individuali ITSC possono essere isolati tra i giorni 21 e 28 giorni di transdifferenziamento. </p><ol><li> Aggiungere 40 ml di soluzione di tripsina / EDTA 0,05% in 12 pozzetti di una ben piatto 96 U-fondo e mettere il piatto sul ghiaccio. </li><li> Prima di isolamento delle colonie, media aspirare a 4F-MEF piatto transdifferenziamento. Lavare le cellule una volta con 10 ml di PBS. Anche in questo caso, aggiungere 10 ml di PBS e posto piatto sotto un microscopio invertito. </li><li> Usando una pipetta da 100 ml insieme a 40 ml sollevamento singolo colonie, dalla cli ircling con la punta della pipetta ed aspirando la colonia galleggiante. Trasferire le cellule di una colonia ciascuno in un pozzetto del preparato a 96 pozzetti piatto. Dopo aver raccolto 12 colonie pongono 96 pozzetti piatto in incubatore a 37 ° C per 5 min. </li><li> Dissociare cellule pipettando su e giù per diverse volte e sospensione cellulare trasferimento in un pozzetto di un piatto 24 pozzetti con preparati 500 ml di mezzo TS-CM (30% fresco TS media + 70% CM + FGF4 / eparina). </li><li> Il giorno seguente sostituire media con media TS-CM fresca per rimuovere le cellule morte. Cambiare mezzo di ogni altro giorno. </li><li> Monitor per l&#39;aspetto delle colonie epiteliali con contorni luminosi (vedi <strong>figura 2).</strong> Una volta appaiono le colonie, la cultura fino al 70% confluenti o per un massimo di una settimana e gradualmente espandere su piatti di grandi dimensioni. D&#39;ora in poi, le cellule cultura come in buona fede TSC, secondo protocolli standard nei media siero contenente o nei mezzi, definiti privi di siero <sup>3,5,11.</sup> </li></ol>

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	<li>Cambuli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+memory+of+the+first+cell+fate+decision+prevents+complete+ES+cell+reprogramming+into+trophoblast.">Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast.</a> <em style='font-style: italic'>Nat commun</em>. <b>5</b>, 5538 (2014).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Pluripotent+Stem+Cells+from+Mouse+Embryonic+and+Adult+Fibroblast+Cultures+by+Defined+Factors.">Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors.</a> <em style='font-style: italic'>Cell</em>. <b>126</b> (4), 663-676 (2006).</li><li>Kubaczka, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Induction+of+Trophoblast+Stem+Cells+from+Murine+Fibroblasts.">Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.</a> <em style='font-style: italic'>Cell Stem Cell</em>. <b>17</b> (5), 557-568 (2015).</li><li>Benchetrit, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Nuclear+Reprogramming+Underlies+Lineage+Conversion+into+Functional+Trophoblast+Stem-like+Cells.">Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells.</a> <em style='font-style: italic'>Cell Stem Cell</em>. <b>17</b> (5), 543-556 (2015).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> <em style='font-style: italic'>Science</em>. <b>282</b> (5396), 2072-2075 (1998).</li><li>Erlebacher, A., Price, K. A., Glimcher, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+mouse+trophoblast+stem+cell+proliferation+by+TGF-beta/activin.">Maintenance of mouse trophoblast stem cell proliferation by TGF-beta/activin.</a> <em style='font-style: italic'>Dev Biol</em>. <b>275</b> (1), 158-169 (2004).</li><li>Hochedlinger, K., Yamada, Y., Beard, C., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+Oct-4+blocks+progenitor-cell+differentiation+and+causes+dysplasia+in+epithelial+tissues.">Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues.</a> <em style='font-style: italic'>Cell</em>. <b>121</b> (3), 465-477 (2005).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> <em style='font-style: italic'>J Vis Exp</em>.  (64), (2012).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+system+for+the+generation+and+study+of+human+induced+pluripotent+stem+cells.">A high-efficiency system for the generation and study of human induced pluripotent stem cells.</a> <em style='font-style: italic'>Cell Stem Cell</em>. <b>3</b> (3), 340-345 (2008).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> <em style='font-style: italic'>J Vis Exp</em>.  (10), e550 (2007).</li><li>Kubaczka, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+Maintenance+of+Murine+Trophoblast+Stem+Cells+under+Defined+Conditions.">Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions.</a> <em style='font-style: italic'>Stem Cell Rep</em>. <b>2</b> (2), 232-242 (2014).</li></ol>

<p class='jove_content'>The authors have nothing to disclose.</p>

<p class="jove_content"> Il 4 fattore (Tfap2c, GATA3, Eomes, ETS2) protocollo transdifferenziazione basato qui presentata offre un metodo affidabile per generare fedelmente convertito iTSCs da fibroblasti embrionali di topo. Inoltre, il metodo è applicabile anche per i fibroblasti coda post-natale, anche se con un calo di efficienza rispetto ai fibroblasti embrionali <sup>3.</sup> In generale, la qualità dei fibroblasti primari è un fattore critico di esito transdifferenziazione e occorre prestare attenzione a utilizzare le cellule di passag...

<p class="jove_content"> Recentemente, uno studio di confronto diversi approcci di topo cellule staminali embrionali di trofoblasto conversione delle cellule staminali ha rivelato che in tutti i sistemi analizzati, la conversione lignaggio è rimasto incompleto. Invece di cellule staminali indotte (trofoblasto) iTSCs cosiddette staminali trofoblasto cellule simili alle cellule sono stati generati conservando un ricordo del destino delle cellule di origine <sup>1.</sup> Qui, abbiamo seguito un approccio diverso di generazione ITSC. Simile a l&#39;induzione diretta di cellule staminali pluripotenti da murine fibroblasti embrionali (MEF) <sup>2,</sup> iTSCs sono stati convertiti direttamente dal tessuto somatica differenziata. In primo luogo, abbiamo identificato 12 fattori che inducono candidati TSC destino quando sovraespresso in MEF. In seguito, i fattori Tfap2c, GATA3, Eomes e ETS2 sono stati identificati per essere necessaria e sufficiente per l&#39;induzione ITSC <sup>3.</sup> Allo stesso tempo, un altro gruppo ha trovato modo indipendente Tfap2c, GATA3 e Eomes essere sufficiente per convertire MEF in iTSCs. Tuttavia, in talestudio, il tempo richiesto per l&#39;espressione del transgene è notevolmente più lungo rispetto al nostro studio, che indica diverse cinetiche di conversione, quando ETS2 è assente dal transdifferenziamento cocktail <sup>4.</sup> </p><p class="jove_content"> Siero fetale bovino convenzionale (FBS) contenente coltura di cellule staminali del trofoblasto blastocisti derivate indotte e si basa sulla presenza di fattori di crescita secreti dal MEF inattivati <sup>​​5,6.</sup> Durante l&#39;induzione ITSC, questi fattori sono forniti da MEF, che non hanno la piena combinazione di transgeni e non sono sottoposti a transdifferenziamento. Tuttavia, una volta singole colonie ITSC sono sub-coltura, richiedono mezzi di comunicazione, che è stato condizionato dal MEF crescita inattivati. Da lì in poi, iTSCs possono essere coltivate e trattati come blastocisti derivate TSC secondo protocolli standard. Da segnalare, in contrasto con Tanaka <em>et al.</em> <sup>5,</sup> abbiamo quotidianamente cultura TSC e iTSCs senza gelatinizzanti piatti di coltura cellulare. </p>

<table><tbody><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-10G</td><td>Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>Chloroquine diphosphate salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C6628-25G</td><td>Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>Polybrene (Hexadimethrine bromide)</td><td>Sigma-Aldrich</td><td>107689-10G</td><td>Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>human recombinant Fibroblast Growth Factor 4</td><td>ReliaTech</td><td>300-131L</td><td>Dissolve in sterile 0.1% BSA in PBS, prepare 25 &amp;micro;g/ml stock solution. Store aliquots at -80 &amp;deg;C</td></tr><tr><td>Heparin sodium salt</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td>Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80&amp;deg;C</td></tr><tr><td>ProFection Mammalian Transfection System</td><td>Promega</td><td>E1200</td><td></td></tr><tr><td>PBS, no calcium, no magnesium</td><td>ThermoFisher Scientific</td><td>1419-094</td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>ThermoFisher Scientific</td><td>31966-021</td><td></td></tr><tr><td>RPMI 1640, no glutamine</td><td>ThermoFisher Scientific</td><td>31870-025</td><td></td></tr><tr><td>Advanced DMEM (1x)</td><td>ThermoFisher Scientific</td><td>12491-015</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>ThermoFisher Scientific</td><td>25300-054</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30071.03IR</td><td></td></tr><tr><td>Surfactant-free cellulose acetate-membrane filter</td><td>Corning</td><td>431220</td><td></td></tr><tr><td>Non-essential amino acids</td><td>ThermoFisher Scientific</td><td>11140-035</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>ThermoFisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Sodium Pyruvate&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11360-039</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>25030-24</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>ThermoFisher Scientific</td><td>31350-010</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO), cell culture grade</td><td>Panreac AppliChem GmbH</td><td>A3672,0100</td><td></td></tr><tr><td>pLV-tetO-Tfap2c</td><td>Addgene</td><td>70269</td><td></td></tr><tr><td>pLV-tetO-Gata3</td><td>Addgene</td><td>70270</td><td></td></tr><tr><td>pLV-tetO-Eomes</td><td>Addgene</td><td>70271</td><td></td></tr><tr><td>pLV-tetO-Ets2</td><td>Addgene</td><td>70272</td><td></td></tr><tr><td>pLV-tetO-mCherry</td><td>Addgene</td><td>70273</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>FUdeltaGW-rtTA&amp;nbsp;</td><td>Addgene</td><td>19780</td><td></td></tr><tr><td>Mice B6.Cg-Gt(ROSA)26Sor&lt;sup&gt;tm1(rtTA*M2)Jae/J 7&lt;/sup&gt;</td><td>JAX Mice</td><td>6965</td><td></td></tr><tr><td>129S2SV</td><td>Charles River</td><td>129</td><td></td></tr></tbody></table>

protocol for the direct conversion of murine embryonic fibroblasts into trophoblast stem cells

<p class="jove_content"> cellule staminali Trophoblast (TSC) sono una conseguenza della prima decisione destino cellulare in sviluppo dei mammiferi. Essi possono essere coltivate <em>in vitro,</em> mantenendo la capacità di auto-rinnovarsi e di differenziarsi in tutti i sottotipi del lineage trofoblasto, equivalenti <em>in vivo</em> staminali popolazione cellulare dando luogo alla parte fetale della placenta. Pertanto, TSC offrono un modello unico per studiare lo sviluppo placentare ed embrionale rispetto a extra-embrionali decisione destino della cellula <em>in vitro.</em> Dal stadio di blastocisti in poi, una barriera epigenetica distinta composto da metilazione del DNA e degli istoni modifiche separa ermeticamente entrambe le linee. Qui, descriviamo un protocollo per superare questa barriera completamente lignaggio da transitori sovra-espressione del trofoblasto regolatori chiave Tfap2c, GATA3, Eomes e ETS2 in fibroblasti embrionali murini. Le cellule staminali del trofoblasto indotte sono in grado di auto-rinnovarsi e sono quasi identiche a blastocisti di cellule staminali derivate trofoblasto in termini di morfologia, l&#39;espressione del gene marcatore e modello di metilazione. Funzionale <em>in vitro</em> e <em>in vivo</em> confermano che queste cellule sono in grado di differenziarsi lungo lignaggio trofoblasto generare cellule giganti poliploide trofoblasto e chimerizing la placenta quando iniettato in blastocisti. L&#39;induzione di cellule staminali dal tessuto trofoblasto somatica apre nuove strade per studiare le caratteristiche genetiche ed epigenetiche di questo lignaggio extra-embrionale e offre la possibilità di generare linee di cellule staminali trofoblasto senza distruggere l&#39;embrione rispettivo. </p>

<p class="jove_content" fo:keep-together.within-page="1"> Sul piatto dove viene attivato espressione del transgene della 4F, cellule cambiano rapidamente morfologia (confrontare <strong>Figura 2A e B).</strong> Intorno giorno 14 - 21 aree distinte transdifferentiated emergono (due esempi sono riportati nella <strong>figura 2B e C).</strong> Queste colonie primarie mancano morfologia tipica TSC; tuttavia una volta che sono sub-coltura, morfologia caratteristica epiteliale con bordi stretti e confini luminosi ricorda molto da vicino TSC in buona ...

Watch this Scientific Journal Video about Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells at JoVE.com

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

<p class='jove_content'>Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.</p>

Protocollo per la conversione diretta dei fibroblasti murini embrionali in trofoblasto cellule staminali

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

protocol-for-direct-conversion-murine-embryonic-fibroblasts-into

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.6K Views.  Institute of Pathology, University of Bonn Medical School. Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Video: Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

Estrazione

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Didattica

JoVE Lab Manual

Chimica

Lab Manual Sub Articles

Esterification

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Esterification<button class="expand">Expand</button>
	<p>In this lab, you will synthesize an ester from a carboxylic acid and an alcohol in the presence of sulfuric acid. This reaction is called Fischer esterification. The sulfuric acid makes the carboxylic acid more reactive towards the alcohol. Without it, esterification would be slow and unfavorable. Esterification is highly reversible, so you&#39;ll use an excess of alcohol to drive the reaction towards the ester.</p>
	<p>You will be assigned one of seven carboxylic acid-alcohol pairs for your reaction. Each of the seven possible product testers has a distinct fruity scent. When you finish the reaction, you will waft your products vapor towards yourself and identify it based on the fruit that it smells like.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you get started, put on a lab coat, safety glasses, and nitrile gloves. <strong>Note: </strong>Your instructor will demonstrate wafting before handing out the reagents. Always smell compounds by wafting, rather than direct inhalation.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Once you are ready to begin, get your assigned carboxylic acid and alcohol and bring the vials to your fume hood. The vial labeled &#8216;A&#8217;, &#8216;B&#8217;, or &#8216;C&#8217; is your carboxylic acid. The other vial is the alcohol. Record the letters of your carboxylic acid and alcohol in your lab notebook.
		<h4><strong>Table 1: Scent of Carboxylic Acid, Alcohol, and Ester</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td><strong>Letter </strong></td>
					<td><strong>Scent </strong></td>
				</tr>
				<tr>
					<td>Assigned carboxylic acid</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Assigned alcohol</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Synthesized ester</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/68/Table_1._Scent_of_carboxylic_acid,_alcohol,_and_ester.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Now, get a piece of filter paper and a Pasteur pipette. In the fume hood, use the Pasteur pipette to apply a drop of your alcohol to the filter paper.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Bring the filter paper to the front of the hood and waft the scent of the alcohol towards yourself. Be careful because it might smell unpleasant.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, put the filter paper back in the hood and write a brief description of the scent of the alcohol in your lab notebook. Do the same for your carboxylic acid if it&#39;s a liquid using a new pipette and piece of filter paper. <strong>Note:</strong> If you were assigned a solid carboxylic acid, write &#39;solid&#39; in your lab notebook and skip this step.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, attach an open 3-prong clamp to a lab stand in your fume hood. Place a lab jack at the base of the stand and set a stir plate and a heating mantle on top of it. Move the temperature controller next to the setup.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Then, place a small stir bar in a 50-mL round-bottom flask and securely clamp the flask over the heating mantle.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add the carboxylic acid to the flask using a funnel. Rinse the carboxylic acid vial into the flask with 1 or 2 mL of your alcohol, and then add the rest of the alcohol to the flask.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Next, bring a 10-mL graduated cylinder to the fume hood reserved for acids, and carefully measure 4 mL of 3 M sulfuric acid. <strong>Note:</strong> It&#39;s highly toxic and corrosive, so be careful with it.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Cover your graduated cylinder with plastic paraffin film. Then, bring the acid back to your hood and add it to the flask. It&#39;s OK if the liquids form two layers.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Now, use one piece of tubing to connect the lower arm of the standard condenser to the water tap in your fume hood. Attach the other piece of tubing to the upper arm and direct it to your drain. Make sure that the connections are secure.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Apply vacuum grease to the lower ground glass joint of the condenser. Fit the condenser into the round-bottom flask and ensure that the grease forms a good seal.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Secure the condenser in place with a second 3-prong clamp and make sure that the tubing is out of the way. Use a laboratory wipe to remove excess grease, and then secure the connection with a joint clip.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Now, slowly open the water tap until water is flowing smoothly through the condenser. Make sure that the water is flowing into the drain and that there are no leaks.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Then, raise the lab jack until the round-bottom flask is sitting in the heating mantle. You are now ready to start heating your reaction mixture to reflux.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Set the stir motor to medium speed and start heating the mixture on medium heat or with a set point of about 110 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">The first boiling point of your mixture will be between 65 and 100 &#176;C. So, keep an eye on it as it heats and adjust the temperature setting as needed.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Once the mixture reaches an even boil, let it reflux for 50 min.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Then, turn off the heat, lower the lab jack, and replace the heating mantle with a round bottom flask holder.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Let the mixture cool to room temperature with the condenser still in place, which usually takes about 10 min. During that time, prepare an ice bath in a 600-mL beaker and bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Once the mixture has cooled completely, turn off the flow of water to the condenser. Carefully separate the condenser from the flask and move it out of the way. Then, raise the ice bath up to the flask and let it cool for 10 min.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">After that, bring a clean 10-mL graduated cylinder to the base hood and obtain 5 mL of saturated sodium bicarbonate.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Back at your hood, use a lab wipe to remove vacuum grease from the inside of the neck of the round bottom flask.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Then, obtain several clean Pasteur pipettes and some pH paper. Use a pipette to slowly add 1 mL of saturated sodium bicarbonate to the flask and stir the mixture with a stir bar or a glass rod.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Add the rest of the sodium bicarbonate solution a milliliter at a time, stirring after each addition.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Then, use a new pipette to check the pH of the solution. If the pH is &#60; 7, get a few more milliliters of sodium bicarbonate and add it 1 mL at a time, stirring and checking the pH after each addition.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once the solution is at a neutral pH, remove the ice bath and dry the outside of the flask with a paper towel.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Let the mixture sit for a few minutes to see whether it settles into two layers.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Then, obtain a clean Pasteur pipette and a new piece of filter paper. Carefully draw up a small amount of the product solution and apply one or two drops to the filter paper. If your product mixture separated, test the top layer first.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Bring the filter paper to the edge of the fume hood and waft the set towards yourself. The seven possible scents are apple, banana, grape, orange, pear, pineapple, and strawberry.</div>
		</li>
		<li data-sub-note-item="1-33">
		<div class="lab-step">Record the fruit that you think your ester smells like in your lab notebook. If you aren&#39;t sure which fruit it is, ask your instructor for the reference scents to help you decide.</div>
		</li>
		<li data-sub-note-item="1-34">
		<div class="lab-step">Once you&#39;ve identified the smell of your ester, retrieve the stir bar, and dispose of your reaction mixture in the non-halogenated organic waste container. Clean your glassware and stir bar using your usual methods.</div>
		</li>
		<li data-sub-note-item="1-35">
		<div class="lab-step">Next, unclamp the condenser and carefully tilt it to let the water drain through the outlet tubing. Disconnect the inlet tubing from the water tap, keeping the end raised to avoid spills, and let the remaining water flow through the condenser to the drain.</div>
		</li>
		<li data-sub-note-item="1-36">
		<div class="lab-step">Then, disconnect the condenser from the tubing and remove the vacuum grease on the bottom joint with lab wipes. Clean the inner walls of the condenser using your usual methods.</div>
		</li>
		<li data-sub-note-item="1-37">
		<div class="lab-step">Leave the filter papers in the hood until they stop smelling strongly of fruit. Dispose of used Pasteur pipettes in the glass waste and throw out any other trash in the lab trash container.</div>
		</li>
		<li data-sub-note-item="1-38">
		<div class="lab-step">Lastly, put away your equipment and clean your hood before you leave.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>Here are the seven possible esters that you could have made and their scents: propyl acetate smells like pear, isoamyl acetate smells like banana, octyl acetate smells like orange, butyl butyrate smells like pineapple, ethyl butyrate smells like strawberry, methyl butyrate smells like apple, and methyl anthranilate smells like grape.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">Compare the scent of your ester to find the corresponding structure. For example, a strong scent of pears means that you made propyl acetate.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Determine your starting carboxylic acid and alcohol. <strong>Note: </strong>Remember that alcohol attacks the carboxylic acid in this reaction. The resulting intermediate rearranges and eliminates a water molecule to form an ester.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Studies have shown that the alkoxy oxygen comes from the alcohol, not the carboxylic acid. So, we&#39;ll split the ester diagram at the carbon-oxygen single bond and fill in the missing OH and H to get the starting materials. For example, if we split propyl acetate at that bond, add an OH to the carbonyl carbon and add a hydrogen to the alkoxy oxygen, we get acetic acid and 1-propanol as the starting materials.</div>
		</li>
	</ul>
	</li>
</ol>

Esterification: Synthesis of Ester by Fischer Esterification - Procedure

Esterificazione

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	EsterificationExpand
	In this lab, you will synthesize an ester from a ...

Evolutionary Relationships

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Forming an Evolutionary Hypothesis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart used to depict the hypothetical genealogical relationships between species. The tips or leaves of the chart represent specific species and the branches of the tree are different lengths. The different lengths represent the degree of change between each of the species. The common ancestor of all of the species that a specific line branches from is located at a node, where the branches intersect. Species that share a node are called a sister group.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, look at the provided blank cladogram and make a hypothesis about where the assigned animals should be placed on the tree. If a blank cladogram has not been provided please <a target="_blank" href="/files/ftp_upload/10562/Blank_Cladogram.png">download one here</a>.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, observe the image of the fossilized animal.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Using the morphology, or physical characteristics, of the fossil, make a hypothesis about where on your cladogram it should be placed. Add a new line or mark to indicate your proposed position for the fossil.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Comparing Gene Sequences Using BLAST
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Historically, cladograms have been constructed through the comparison of morphology. Now, genetic sequences can also be compared between species of interest to construct cladograms. DNA sequences are not usually preserved in fossils, so for this lab you will use the BLAST database to compare the DNA sequences of living species that are closely related to the fossil to thousands of other living species.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Before beginning this lab, your instructor will have downloaded the three preserved gene sequences to a folder on your desktop.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Navigate to the BLAST homepage, which the instructor should have left open in your browser, and click on Saved Strategies from the menu at the top of the page.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Click Open on the Evolutionary_Relationships_Gene1.txt file.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, click View to go to the Standard Nucleotide BLAST search page. You should be able to see the DNA sequence in the Query Sequence box at the top-right of the screen.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Scroll to the bottom of the screen and click the BLAST button. Note: It will take a few moments to process and analyze the data. 
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the data has been processed, a graphic summary will appear.  NOTE: Your query sequence is represented by the blue line at the top of the box. Each of the lines below the blue line represents another sequence in the BLAST database that matches your query. The length and location of these bars represent where and how much of the sequence match up to the query. The color of the bar represents its score, or how identical the sequence is to the query. Below the graphic summary is the Sequences producing significant alignments list, which contains descriptions of the DNA sequences retrieved from the database that most closely align with the query sentence. These are listed in descending order, from the most similar at the top to the least similar at the bottom. On the right, there are several statistics on how closely related each database sequence is to the experimental sequence. The higher the Max, Total, Query Coverage, and Identity scores are, the more similar the query and retrieved sequences are to each other. Similarly, the lower the E value number, the less likely the sequences match was found by random chance and more significantly the alignment is accurate. An E value of 0 indicates a highly significant match.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Click on the name of the description of the most similar alignment listed. This will take you further down the page to the exact DNA sequence alignment between the retrieved sequence and the query sequence.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Click on the Sequence ID number. This will open a new tab with more specific information on the retrieved gene sequence.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Identify and record the scientific and common names of the organism, which should be listed next to Source.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Identify and record the protein that the gene codes for, which should be listed next to Definition.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Close the tab and hit Back to return to the Sequences producing significant alignments list.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Then, repeat steps 7 &#8211; 9 for the next most similar sequences.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">After collecting several species names, return to the Sequences producing significant alignments list and click Select: All to check all of the boxes next to the names of each listed alignment sequence.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Click on Distance of tree results to create a phylogram based on the similarity of all of the BLAST result gene sequences to the query gene sequence.  Note: Your query fossil relative sequence will be highlighted in yellow.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">Record your sequence&#8217;s location in relation to the other taxon groups shown in the tree.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Finally, return to the Saved Strategies tab and upload the next saved gene sequence.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">Query the remaining two fossil relative DNA sequences as previously demonstrated (steps 1 &#8211; 16).
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Based on your gene sequence results, come up with a second hypothesis about where your fossil specimen fit in the original cladogram.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Compare this placement with your first hypothesis.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Compare your tree with your classmates&#8217;. If any of your classmates put the fossil on a different branch, ask them how they came to that decision.
</div></li></ul></li></ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Procedure

Relazioni evolutive

Forming an Evolutionary Hypothesis
ExpandNOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart ...

Biologia

Evoluzione

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse=""><strong>Bacterial Transformation</strong>
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">IMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face away from suspension cultures, and to avoid inhaling reagents. Do not touch your face while performing the experiment. Always wash your hands before and after every experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">When you are ready to safely begin, check to make sure you have a tube or tubes of competent <em>E. coli</em> cells in your ice bucket.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, transfer 50 &#181;L from one of these tubes to each of two new micro-centrifuge tubes, one labeled '&#8211;', and one labeled '+'.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, pipet 2 &#181;L of p-GREEN plasmid into the plus tube and flick the tube gently to incorporate the plasmid throughout the bacterial solution. HYPOTHESES: The experimental hypothesis is that on plates containing no antibiotic, bacteria with or without the plasmid can survive, and so both will grow lawns of bacteria. Conversely, on the plates containing the ampicillin antibiotic, only the bacteria successfully transformed with the resistance plasmid will grow, whereas the minus plasmid plates will contain no bacteria. The null hypothesis of this experiment is that there will be no difference in bacterial growth between the plates.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Incubate both tubes on ice.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After 30 minutes submerge both tubes 3/4 of the way into the water of a 42 &#176;C water bath for 30 seconds.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">At the end of the water bath incubation, place the tubes back on ice for 5minutes, then add 950 &#956;L of room temperature SOC media to each tube.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Incubate the tubes in a shaker at 37 &#176;C for 60 minutes at 250 rpm.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">While the tubes are incubating, label the bottoms of one LB and one LB/ampicillin, or AMP plate, &#8220;+ plasmid&#8221;, and the bottoms of one LB plate and one LB/AMP plate &#8220;- plasmid&#8221;.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">At the end of the incubation, pipet 50 &#956;L from the &#8220;+ plasmid&#8221; cell tube onto both the LB/AMP plus plate and the LB plus plate.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Then, with a fresh tip, add 50 &#956;L of the bacteria that were not exposed to the plasmid to each of the minus plates.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, spread each plate using a fresh pipette tip.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Cover the plates with their lids, and then seal them with laboratory film.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then store the plates upside down in a 37 &#176;C incubator for 24 hours.
</div></li></ul></li><li data-note-item="2" data-parent-collapse=""><strong>Results</strong>
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">The day after the transformation, observe which plates have bacteria, and how the bacteria are distributed over each plate.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Record the number of colonies and the color of the colonies for different conditions. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/11/Table_1_Bacterial_Transformation.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To determine the initial mass, IM, of the plasmid, first multiply the plasmid concentration from the stock tube of p-GREEN by the volume transferred into the experimental tube.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate how much DNA is spread onto each plate, divide the volume of bacterial suspension spread onto each plate by the volume of the suspension in the tube, and multiply this fraction by the initial mass of plasmid used.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">To determine the transformation efficiency, count all of the visibly distinct colonies on the plus plasmid LB/AMP plate, and divide this number by the mass of plasmid spread. NOTE: The unit for transformation efficiencies is cfu/&#956;g of plasmid DNA.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Record all of the results for each plate in the table and analyze them.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Observe any differences between the colonies and make hypotheses to explain this observation. Consider the factors that may affect the transformation efficiency of competent bacteria.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">NOTE: You will have also noticed that each plate had a different outcome. The minus plasmid and plus plasmid LB plates should have grown a lot of bacteria across their plates. The minus plasmid LB/AMP plate should have no bacterial growth. And the plus plasmid LB/AMP plate should have grown bacterial colonies that fluoresced green.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Procedure

Trasformazione batterica

Bacterial Transformation
ExpandIMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face ...

Genetica

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

Struttura cellulare

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

Fondamenti

Population Growth

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Exponential Population Growth Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In these experiments you will use computer software to simulate different types of growth models. HYPOTHESES: The experimental hypothesis might be that if we increase either R, the reproductive rate or the initial population size, NT, that this will increase the population size after 10 generations in an exponential growth model compared to unmanipulated control populations. The null hypothesis may be that populations of different R and initial population size will have the same population size as control groups after 10 generations.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To build an exponential population growth model, first open a new workbook in Excel.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Save the file to a secure location and name it &#8220;exponential growth&#8221;.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Label cell A1 &#8220;generation&#8221;, and cell B1 &#8220;population size&#8221;.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Type the number zero into cell A2. This row will represent the initial generation.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">In cell A3 type this formula: = 1 + A2
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Click on cell A3 and drag the small box in the bottom right corner to cell A12 for 10 generations.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Type the number two in cell B2. This will represent the initial population size in the model.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label cell E1 &#8220;growth rate&#8221;, and in cell E2 type the number 0.05. This value represents the proportion of new offspring produced by each individual for each generation, or R in the exponential growth model.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">NOTE: In the classic exponential growth equation, T is the generation, and NT is the number of organisms in the current generation. NT+1 will be is the number of individuals in the next generation.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Enter the exponential growth equation in cell B3 as the formula shown here: = B2 + (1 + E2) * B2
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, hit enter.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Click cell B3 and drag the small square in the bottom right corner down to cell B12 to simulate 10 generations of exponential growth.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To observe the results, select all data, along with the headers, in columns A and B.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Click &#8220;insert&#8221; and select &#8220;scatter plot with smooth lines and markers&#8221; from the toolbar. A scatter plot should appear in the sheet. TIP: If it does not, check to see if a new sheet was created in the workbook.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Label the Y axis &#8220;population size&#8221; and the X axis &#8220;generation&#8221;. Title the chart &#8220;exponential growth of a population&#8221;.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Observe how the population increases in a classic exponential growth pattern.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Next, create Table 1 labeling the &#8220;growth rate R&#8221; as the header of column A, and &#8220;population size after 10 generations&#8221; as the second column.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Record the population size at generation 10 for the growth rate of 0.05, the rate we just simulated.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, set the growth rate in cell E2 to 0.1, and repeat the steps to generate a new scatter plot.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Record the population size at generation 10.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Continue to alter the growth rate using the values in table one, noticing how the Y axis changes to accommodate the increasing values.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">After completing Table 1, set the growth rate back to 0.05 and change the initial population size in cell B2 to 100 individuals.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Record the population size at generation 10 in a new table, Table 2.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Proceed to change the initial population size using the values in Table 2. Once the table is complete, save the Excel sheet and close it.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Logistic Population Growth Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">HYPOTHESES: In a logistic growth model, the experimental hypothesis could be that populations will approach and meet the carrying capacity, and any populations with sizes greater than the carrying capacity will decrease in the subsequent generation. The null hypothesis might be that population growth will not follow a logistic S-shaped curve in the logistic models, and that generations with population sizes greater than the carrying capacity will continue to increase or stay the same in the next generation.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">To build a logistic population growth model, open a new spreadsheet in Excel.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Save the file as &#8220;logistic growth&#8221; in the same folder as before.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Then label cell A1 &#8220;generation&#8221; and B1 &#8220;population size&#8221;.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Type the number zero in cell A2. This row will represent the initial generation.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">In cell A3 type the formula: = 1 + A2
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Now, hit enter.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Now, click on cell A3 and drag the small box in the bottom right corner to cell A12 to number up to the 10th generation.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Type the number two in cell B2 to represent the initial population size.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Label cell E1 as &#8220;max growth rate&#8221; and cell F1 as &#8220;carrying capacity&#8221;.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Finally, set the max growth rate in cell E2 to 0.05. This value will represent the maximum growth rate the population may achieve &#8211; &#8220;R max&#8221; in the discrete logistic equation. NOTE: In the classic logistic growth equation the term K represents carrying capacity. As the population size of the current generation or NT, approaches the carrying capacity, the growth of the population begins to slow.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Now set the carrying capacity in cell F2 to 50.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Enter the logistic growth equation in cell B3 as the formula below: = B2 + ((1 + E$2) * B2) * ((F$2 - B2) /F$2)
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Drag the formula down to cell B12 to add it to all 10 generations.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">To observe the results of this exercise, highlight all data and headers in columns A and B, then click &#8220;insert&#8221; and select &#8220;scatter plot with smooth lines and markers&#8221;.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Label the Y axis population size and the X axis generation.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">Title the chart as logistic growth of a population.
</div></li><li data-sub-note-item="2-19"><div class="lab-step">Observe the growth of the population as it nears carrying capacity and record the generation that the population meets or exceeds carrying capacity in a new table.
</div></li><li data-sub-note-item="2-20"><div class="lab-step">Now set the max growth rate in cell E2 to 1.
</div></li><li data-sub-note-item="2-21"><div class="lab-step">Notice how the population responds when it rises over carrying capacity.
</div></li><li data-sub-note-item="2-22"><div class="lab-step">Continue to set the max growth rate to the values in Table 3 and record the generation at which the population rises two or above carrying capacity.
</div></li><li data-sub-note-item="2-23"><div class="lab-step">Now set the max growth rate back to 0.05 and set the carrying capacity to 1,000.
</div></li><li data-sub-note-item="2-24"><div class="lab-step">Notice how the graph changes as the growth approaches the carrying capacity.
</div></li><li data-sub-note-item="2-25"><div class="lab-step">Drag the formulas in A12 and B12 down to A14 and B14.
</div></li><li data-sub-note-item="2-26"><div class="lab-step">Adjust the plot to include these new data points to show when the population reaches carrying capacity.
</div></li><li data-sub-note-item="2-27"><div class="lab-step">Save the Excel sheet and close it.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Predator-Prey Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Hypotheses: In our third simulation of predator-prey logistics, the experimental hypothesis could be that predator populations will increase as prey populations increase and that the two populations will experience growth and decline dependent on the population of the other species. The null hypothesis may be that the two populations will show patterns of growth unrelated to the other species&#39; population size.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">To build a predator-prey model, open a new spreadsheet in Excel.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Save the file in the same folder as the other two models and name it &#8220;predator-prey model&#8221;.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Label cell A1 &#8220;time&#8221;, cell B1 &#8220;prey&#8221; and cell C1 &#8220;predator&#8221;.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">In cell A2 type zero and hit enter.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">In cell A3 type the formula below: = 1 + A2
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Drag the formula down to cell A802 as the model will require many time points to visualize changes of both populations.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Now type 100 into cell B2 to represent the initial prey population and type 25 into cell C2 as the initial predator population.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Label cell E1 &#8220;prey growth rate&#8221;, cell F1 &#8220;consumption of prey by predator&#8221;, cell G1 &#8220;conversion efficiency&#8221; and cell H1 &#8220;starvation of predator&#8221;.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">Type the number 0.02 in cell E2. This value represents the growth rate of the prey species or R in the prey population model. NOTE: Here the prey population is represented as VT and the predator population as CT. The value A is the per capita attack rate of the predator species on the prey. In the predator population model, variables are introduced for predators produced per prey consumed, F or conversion efficiency as well as for the starvation rate of the predators noted as Q.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Type 0.0005 in cell F2 for the attack rate in both the prey population and the predator population model.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Then type 0.8 in cell G2 to represent the conversion efficiency, and 0.05 in cell H2 for the starvation rate in the predator population equation.
</div></li><li data-sub-note-item="3-14"><div class="lab-step">Now type the prey growth equation in cell B3 as the formula below: =B2+(E$2*B2)-(F$2*C2*B2)
</div></li><li data-sub-note-item="3-15"><div class="lab-step">Hit enter.
</div></li><li data-sub-note-item="3-16"><div class="lab-step">Then type the prey growth equation in cell C3 as the displayed formula: =C2 + (F$2*G$2*B2*C2)-(H$2*C2)
</div></li><li data-sub-note-item="3-17"><div class="lab-step">Hit enter.
</div></li><li data-sub-note-item="3-18"><div class="lab-step">Hold down the shift keys and click to select both cells B3 and C3.
</div></li><li data-sub-note-item="3-19"><div class="lab-step">Click the small box at the bottom right corner of cell B3 and drag the two formulas down to row 802 to simulate the predator and prey populations over 800 generations.
</div></li><li data-sub-note-item="3-20"><div class="lab-step">To visualize the data, select all of the data and headers in columns A, B and C.
</div></li><li data-sub-note-item="3-21"><div class="lab-step">Click &#8220;insert&#8221; and select &#8220;scatter plot with smoothed lines&#8221;.
</div></li><li data-sub-note-item="3-22"><div class="lab-step">A chart showing the population change of the predator and the prey populations should appear.
</div></li><li data-sub-note-item="3-23"><div class="lab-step">Label the Y axis of the plot population size and the X axis time.
</div></li><li data-sub-note-item="3-24"><div class="lab-step">Title the plot &#8220;predator-prey interactions&#8221;.
</div></li><li data-sub-note-item="3-25"><div class="lab-step">Observe the interactions between the predator and prey populations.
</div></li><li data-sub-note-item="3-26"><div class="lab-step">Set the initial predator population to zero and observe what happens in the plot.
</div></li><li data-sub-note-item="3-27"><div class="lab-step">Now set the initial predator population to 120 and observe what happens to the predator population when there are more predators than prey.
</div></li><li data-sub-note-item="3-28"><div class="lab-step">Finally set the initial prey population to zero and observe what happens to the population of the predators.
</div></li><li data-sub-note-item="3-29"><div class="lab-step">Experiment with other values to see what other interactions can be created.
</div></li><li data-sub-note-item="3-30"><div class="lab-step">When finished, save the workbook and close it.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">In the exponential growth model built in the first activity, observe the shape of the resulting population growth curve, and consider whether you think this type of growth could be sustained in nature.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Note how the growth rate R affected the population size after 10 generations in the exponential growth model.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Also consider how the initial population size affected the population size after 10 generations in the exponential growth model.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Now, consider the logistic growth model, and observe the shape of this graph, noting how it differs from the exponential growth model.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Then, note how the populations behave as they approach the carrying capacity in your logistic growth model.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Decide if you think that the maximum per capita growth rate, R max, influenced how quickly populations reached carrying capacity in the logistic growth model.
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Also, consider what happens to population size when populations are over carrying capacity in the logistic growth model, or what might happen if the carrying capacity is lowered.
</div></li><li data-sub-note-item="4-8"><div class="lab-step">In the predator-prey model, note whether the prey population responded to increasing predator population. Conversely, consider how the predators then respond to decreasing prey populations.
</div></li></ul></li></ol>

Population Growth: Exponential and Logistic Growth and Predator-Prey Model - Procedure

Crescita della popolazione

Exponential Population Growth Model
ExpandNOTE: In these experiments you will use computer software to simulate different types of growth models. ...

Ecologia

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://jove.unicartagenaproxy.elogim.com/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

Diversità microbica e fungina

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be labeled with the notation REF for red-eyed flies or WEF for white-eyed flies. The female vial should also be labeled virgin as these flies should be unmated for the purpose of this experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Make sure you have around five flies in each vial. NOTE: You will perform crosses using these flies to determine if red or white eyes is dominant and if the trait is sex-linked. You should also have two fly anesthesia wands, a paint brush, magnifying glass, fly anesthetic, some empty fly vials, and a sheet of white paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Draw a line in the middle of the paper to make a male side and a female side.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Dip the fly anesthesia wand into the solution and insert it into the red-eyed female vial until the flies stop moving.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, dip the same wand into the white-eyed male vial until these flies are also anesthetized.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Tap the flies from the two vials onto the sheet - Females on one side and males on the other.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Check to make sure that all the flies from the female vial are female and likewise, confirm that the male flies are male.  NOTE: Males are slightly smaller and have a darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If there are any male flies in the female vial, obtain a new vial as these females likely have sperm from males from their own strain and are unusable.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Now, move the five red-eyed females and five white-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label this vial to indicate that this is a cross between red-eyed females and white-eyed males. HYPOTHESES: Use Punnett squares to formulate hypotheses for this experiment. Consider what you will see in a few days if the white allele is dominant and there is no effect of parental sex. What if red is dominant? Under the potential offspring genotype in each box, write the expected phenotype.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">To set up the reverse cross, again dip the fly anesthesia wand into the solution and insert it this time into the white-eyed female vial and then the red-eyed male vial until all the flies have stopped moving.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Tap the flies onto a sheet of white paper and check to make sure that all the flies from the female vial are female and likewise with the male flies.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, move the five white-eyed females and five red-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Label this vial to indicate that this is a cross between white-eyed females and red-eyed males.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, place the two tubes containing the new crosses into a rack for incubation at room temperature. NOTE: After about three days, larvae should be moving around in the vial.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Remove the 10 adult flies and dispose of them into the morgue tube containing 70% ethanol.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, put the tubes back in the rack and let the larvae mature until they reach the adult stage in up to two weeks.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Once all of the larvae from these crosses have matured to adulthood, anesthetize the flies in one of the tubes using the wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, on a piece of white paper, sort the flies by sex and phenotype and then record these data.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Now, repeat this process and tally the sexes and phenotypes for the second cross (steps 1 &#8211; 18).
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Finally, once you have counted all of your flies, dispose of them in the morgue vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin investigating your results, first compare the data you collected with the hypothesis you made using the Punnett squares.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Did your data match the simple Punnett square hypothesis where red or white is dominant and there is no effect of sex? Compare your data with other lab groups.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">If your data does not match what your Punnett squares predicted, does this indicate that the gene may be sex-linked?
</div></li><li data-sub-note-item="2-4"><div class="lab-step">If the red gene were dominant and sex-linked, when we cross red-eyed females with white-eyed males, all of the offspring would have red eyes. Assuming all of the females were homozygous for the color allele, does this match your data for the first cross?
</div></li><li data-sub-note-item="2-5"><div class="lab-step">If white were dominant and the gene were sex-linked, we would see the exact opposite. So all of the flies would have white eyes. From your data, can you conclude from your first cross whether red or white is dominant?
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When the reciprocal cross of white-eyed females and red-eyed males is performed, if the red phenotype is dominant, all of the females would be red and all of the males would be white. This would also confirm that the gene is specifically x-linked because white-eyed females could then only be produced in crosses with white-eyed males. Did your second cross produce any white-eyed females?
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Finally, if the gene were x-linked and white was dominant, crossing white-eyed females with red-eyed males would result in all of the offspring having white eyes. Does this hypothesis fit with your second cross?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Look over your data and conclude which of the hypotheses best fits the patterns you found in your crosses. If your data does not fit with any of these, hypothesize why this might be the case.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Procedure

Genetica degli organismi

REF and WEF Fly Genetics
ExpandNOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Prep

Divisione cellulare

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

Processi cellulari

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse=""><em>D. Melanogaster</em> Care
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Obtain the <em>Drosophila melanogaster</em> specimens at least two weeks before the experiment to allow the specimens to acclimate to the lab room conditions.  NOTE: 25 <em>Drosophila</em> adults produce about 50 new adults in two weeks. Each colony-housing container should consist of a clean vial, 1.5 &#8211; 3 cm of prepared food medium, and a stopper.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a funnel to transfer the flies to the colony-housing container.  NOTE: House the colonies so there will be approximately 40 flies per student or student group (plus spares) on the day of the experiment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">On the day of the experiment, place a dish of apple cider vinegar, a razor blade, a dish of distilled water, two sterile cotton balls, a funnel, scissors, Sharpie pens, a timer, four water bottle caps, and two water bottles onto each lab table.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After the equipment is in place, use a funnel to randomly move colonies of 40 adult flies each into the clean vials without food or substrate.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">NOTE: Vials without food or substrate make it easier for the students to collect only the adults and to avoid adding food to the experimental chambers.
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Prep

Comportamento animale

D. Melanogaster Care
ExpandObtain the Drosophila melanogaster specimens at least two weeks before the experiment to allow the specimens to acclimate to ...