Wir danken Stephan Wilkens für Hilfe in Transmissions-Elektronenmikroskopie. Diese Arbeit wurde von den National Institutes of Health Grants R01AG023731 unterstützt.

Frühere Studien haben gezeigt, dass die ersten 22 aminoterminalen Reste von Mgm101 nach dem Import in Mitochondrien 19 gespalten werden. Zur Expression in Escherichia coli, wird das MGM101 offenen Leserahmen ohne die ersten 22 Codons durch PCR amplifiziert und hinter die männlichen Sequenz, die das Maltose-bindende Protein (MBP) in einer modifizierten Version des Expressionsvektors pMAL-C2E platziert. Dies erzeugt die MBP-Mgm101 Fusion mit einem Linker, der eine Spaltstelle für die Protease PreScission (Abbildung 3). Das Plasmid wird zunächst durch Auswahl E. konstruiert coli Transformanten ohne IPTG und X-Gal blau / weiß-Selektion. Das resultierende Plasmid pMAL-C2E-MGM101 wird dann in den E. eingeführt coli-Stamm BL21-CodonPlus (DE3)-RIL indem Sie Ampicillin und Chloramphenicol-resistenten Kolonien. 1. Expression, Induktion, Cell Lysis und DNase I Behandlung <ol><li> InocUlate frisch Transformanten in 10 ml LB-Medium (1% Bacto Trypton, 0,5% Hefeextrakt, 1% NaCl) mit Glucose (0,2%), Ampicillin (100 ug / ml) und Chloramphenicol (50 ug / ml) ergänzt. Bei 37 ° CO / N unter Schütteln bei 200 rpm inkubiert. </li><li> Impfen die 10 ml Vorkultur in 2 Liter des ergänzten LB-Medium wie oben. Wachsen die Zellen bei 37 ° C unter Schütteln bis OD 600 erreicht 0,5. </li><li> Induzieren der Expression von MBP-Mgm101 Fusionsproteins durch Zugabe von Isopropyl-β-D-1-thiogalactopyranosid (IPTG) in einer Endkonzentration von 0,3 mM. Wachsen die Zellen unter Schütteln bei 30 ° C für 5 Stunden. </li><li> Sammeln der Zellen durch Zentrifugation unter Verwendung eines Beckman JA-10-Rotor (5.500 × g, 4 ° C, 10 min). Nachdem der Überstand verworfen, das Zellpellet in 30 ml Lysepuffer (20 mM Tris-HCl, pH 7,4 und 1 mM EDTA, pH 7,4), der Protease-Inhibitoren (25 uM Leupeptin, 1 uM Pepstatin A und 1 mM PMSF). </li><li> Beschallen Zellsuspension auf Eis für 20sec mit einem Ultraschall-Prozessor (Heat Systems; Modell W-385) bei 50% ED, ermöglichen, sich auf Eis für 30 sec abkühlen und 2x wiederholen. </li><li> 1 ml DNAse 1 stock bei 2 mg / ml. Rock the Zelllysat bei 4 ° C für 2 Stunden. </li><li> Stellen NaCl bis zu einer Endkonzentration von 500 mM. </li><li> Entfernen der Zelltrümmer durch Zentrifugation bei 10.000 × g bei 4 ° C für 30 min. </li></ol> 2. Reinigung mit Amylose-Affinitätschromatographie <ol><li> Äquilibrieren 1,5 ml Amylose-Harz (50% Suspension) in dem Lysepuffer. Fügen Sie das Gleichgewicht Amyloseharz dem Zelllysat. Schaukeln sanft bei 4 ° CO / N. </li><li> Legen Sie das Lysat-Harz-Mischung auf einer Econo-Column Chromatographie-Säule in einem kalten Raum installiert. Lassen Sie die ungebundenen Proteine, um die Spalte durch die Schwerkraft passieren. </li><li> Waschen der Amylose-Harz mit 300 ml Waschpuffer I (20 mM Tris-HCl, pH 7,4, 400 mM NaCl, 1 mM EDTA, pH 7,4 und 0,2 mM PMSF). </li><li> Wiederholen Sie die Wäsche mit 150 ml Waschpuffer II (20 mM Tris-HCl, pH 7,4, 200 mM NaCl, 1 mM EDTA, pH 7,4 und 0,2 mM PMSF). </li><li> 5 ml Elutionspuffer (20 mM Tris-HCl, pH 7,4, 200 mM NaCl, 1 mM EDTA, pH 7,4 und 0,2 mM PMSF, 10 mM Maltose) auf die Säule bei 4 ° C inkubiert für 10 Minuten, Sammeln das Eluat und wiederholen Sie für weitere Male. </li><li> Kombinieren der Eluate, bestimmen die Ausbeute und Reinheit der MBP-Mgm101 durch Laden eines Aliquots des Eluats auf einem SDS-PAGE-Gel von Coomassie-Färbung (Abbildung 4). </li></ol> 3. PreScission Proteasespaltung und Kationenaustausch-Chromatographie <ol><li> In 50 Einheiten PreScission Protease an die MBP-Mgm101 Eluat. Führen die Spaltung bei 4 ° CO / N und lassen es während der Dialyse gegen Dialysepuffer weiter (20 mM Tris-HCl, pH 7,4; 100 mM NaCl; 2 mM DTT, 1 mM EDTA, pH 7,4 und 0,2 mM PMSF) </li><li> Prüfen Sie die Effizienz der Spaltung auf einem SDS-PAGE-Gel (Abbildung 5A). </li><li> Legen Sie das gespaltene MBP-Mgm101 von mehreren injizierenIonen auf einem Bio-Maßstab Mini Macro-Prep Hohe S Patrone zu einem Bio-Rad Biologic DuoFlow FPLC-System verbunden. </li><li> Starten Sie den Kationenaustauscherchromatographie durch Anlegen einer Schritt Salzgradient von 5 mm, 300 mm, 500 mm, 750 mm und 1000 mM NaCl, die durch Mischen von Puffer A (5 mM NaCl erstellt wird; 10 mM Na-Phosphat, pH 7,2; 1 mM PMSF) und Puffer B (1 M NaCl; 10 mM Na-Phosphat, pH 7,2; 1 mM PMSF). Stellen Sie den Durchfluss bei 0,5 ml / min. Sammeln Sie die Mgm101 Fraktion und überprüfen Sie die Reinheit des Proteins auf einem SDS-PAGE-Gel (Abbildung 5B). </li></ol> 4. Size Exclusion Chromatography <ol><li> Kombinieren Sie die Proteinfraktionen aus Kationenaustauscherchromatographie. Dialyse des Proteins in GF Äquilibrierungspuffer (20 mM MOPS, pH 7,0; 150 mM NaCl; 1 mM EDTA, pH 7,4, 5 mM 2-Mercaptoethanol, 0,2 mM PMSF). </li><li> Konzentrieren des Proteins mit VIVASPIN 15R Ultrafiltration Spin-Säule, um die Lautstärke zu ~ 1 ml durch Spinnen bei 3.000 xg bei 4 ° C reduzieren </li><li> Legen Mgm101 ona kalibriert Superose 6 prep Grad Säule, die mit dem Chromatographie-Puffer (20 mM MOPS, pH 7,0; 150 mM NaCl; 5 mM β-Mercaptoethanol, 1 mM EDTA, pH 7,4, 0,2 mM PMSF). </li><li> Starten des Größenausschluss-Chromatographie mit dem Puffer bei einer Flussrate von 0,5 ml / min ausgeführt. </li><li> Sammeln Sie die Fraktionen der gereinigten Mgm101 Gipfeln. Legen Aliquots der Fraktionen auf einem 12% SDS-PAGE zur Überprüfung der Qualität des fertigen Protein. </li><li> Konzentrieren des Proteins mit VIVASPIN 6, dann schnell frieren die Proben in flüssigem N2 und bei -80 ° C </li><li> Verwenden Sie die Mgm101 Proben innerhalb von 6-12 Monaten für ssDNA-Bindungs-Assay (Abbildung 8) und für strukturelle Visualisierung mittels Transmissions-Elektronenmikroskopie (Abbildung 9). </li></ol>

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U.S.A. 96, 4279-4284 (1999).</li><li>Ploquin, M., 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=Functional+and+structural+basis+for+a+bacteriophage+homolog+of+human+RAD52.">Functional and structural basis for a bacteriophage homolog of human RAD52.</a> Curr. Biol. 18, 1142-1146 (2008).</li><li>Chen, X. J., Guan, M. X., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGM101,+a+nuclear+gene+involved+in+maintenance+of+the+mitochondrial+genome+in+Saccharomyces+cerevisiae.">MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.</a> Nucl. Acids Res. 21, 3473-3477 (1993).</li><li>Meeusen, S., 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=Mgm101p+is+a+novel+component+of+the+mitochondrial+nucleoid+that+binds+DNA+and+is+required+for+the+repair+of+oxidatively+damaged+mitochondrial+DNA.">Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.</a> J. Cell Biol. 145, 291-304 (1999).</li><li>Mbantenkhu, M., 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=Mgm101+is+a+Rad52-related+protein+required+for+mitochondrial+DNA+recombination.">Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.</a> J. Biol. Chem. 286, 42360-42370 (2011).</li><li>Nardozzi, J. D., Wang, X., Mbantenkhu, M., Wilkens, S., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., 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=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, S., 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+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Es war eine Herausforderung, um eine stabile, nativen rekombinanten Protein aus E. Mgm101 produzieren coli möglicherweise aufgrund seiner Unlöslichkeit in Bakterienzellen. In diesem Bericht zeigen wir, dass die MBP-Fusion-Strategie das Protein zu einem angemessen hohen Niveau exprimiert werden können. Durch negative Färbung Transmissionselektronenmikroskopie und Größenausschlusschromatographie, haben wir bereits gezeigt, dass die MBP-Fusionsprotein einheitliche Oligomere bildet in vitro ...

Homologe Rekombination ist für die Reparatur von Doppelstrangbrüchen (DSB) und Interstrang Vernetzungen und für die Wiederaufnahme der DNA-Replikation von eingestürzten Replikationsgabeln 1 wichtig. In konventionellen homologen Rekombination ist die zentrale Reaktion der ATP-abhängigen Rekombinasen wie RecA in Prokaryoten und Rad51 und Dmc1 in Eukaryonten 1-3 katalysiert wird. Diese Rekombinase bilden nucleoprotein Filamente auf ssDNA, die unerlässlich für die Einleitung Homologiesuche und Stranginvasion innerhalb duplex DNA-Templates (Abbildung 1, links) 4-7 sind. Zusätzlich zu den herkömmlichen Schema kann homologe Rekombination auch in einer Weise RecA/Rad51-independent (Abbildung 1, rechts). Zum Beispiel kann die Hefe Rad52 und Rad59 Proteine ​​direkt katalysieren die Anlagerung komplementäre ssDNA-Stränge, die durch Resektion von dsDNA Unterbrechungen ausgesetzt sind. Diese Rekombination Prozess, da singen bekanntle Banddurchlaufanlagen, hat in der Regel nicht um homologe Paarung mit dsDNA Vorlagen. Nach dem Glühen werden heterologe Schwänze durch Exonucleasen entfernt und Kerben sind ligiert Genom Kontinuität 8-10 wiederherzustellen. Reparatur durch den einzigen Banddurchlaufanlagen Mechanismus wird oft durch Deletionen von genomischen Sequenzen zwischen direkt wiederholt Regionen begleitet. Rad52 gehört zu einer vielfältigen Gruppe von Proteinen, die Rekombination verbreitet unter 11 Bakteriophagen sind. Diese Proteine ​​sind auch als Single Strand Annealing Proteine ​​(SSAPs) bekannt, auf der Grundlage ihrer Aktivität in der Förderung der Anlagerung homologen einzelsträngige DNA-Moleküle. Die am besten charakterisierten Bakteriophagen SSAPs sind Redβ und Erf vom Bakteriophagen λ und P22, RecT vom Prophagen rac und der Sak-Protein aus der Lactococcus-Phagen UL36. Die SSAPs sind strukturell durch eine typische β-β-β-α fach dadurch, obwohl Ähnlichkeit praktisch Nichterfassungssignal wirdkönnen in ihrer primären Sequenzen. Sie alle bilden große homooligomeren Ringe von 10 - 14 fache Symmetrie in vitro 12-14. Die funktionellen Auswirkungen dieser Eigenschaft höherer Ordnung strukturelle Organisation ist nicht gut verstanden. Wir sind daran interessiert, zu verstehen, den Mechanismus der homologen Rekombination in der mitochondrialen Genoms. Wir haben zuvor die MGM101 Gen, das für die Aufrechterhaltung der mtDNA in Saccharomyces cerevisiae 15 identifiziert. MGM101 anschließend wurde festgestellt, dass mitochondriale Nukleoide an und ist für die Toleranz der mtDNA auf DNA-schädigende Mittel 16 erforderlich. Allerdings wurde in der Studie von Mgm101 wurde zurück in den letzten zehn Jahren durch die Schwierigkeit gehalten rekombinanten Mgm101 erzeugen. Wir haben vor kurzem in der Herstellung von löslichen Mgm101 bei großen Mengen aus E. gelungen coli mit dem MBP-Fusion-Strategie. Dies hat es uns ermöglicht, dass Mgm101 Aktien demonstrierenbiochemische und strukturelle Ähnlichkeiten mit dem Rad52-Familie von Proteinen 17,18. In diesem Bericht wird eine dreistufige Reinigungsverfahren beschrieben, die produziert homogene Mgm101for biochemische und strukturelle Analysen (Abbildung 2).

<table><tbody><tr><td>Expression vector pMAL-c2E</td><td>New England Biolabs</td><td>#N8066S</td><td></td></tr><tr><td>PreScission Protease</td><td>GE Healthcare Life Sciences</td><td>#27-0843-01</td><td></td></tr><tr><td>BL21-CodonPlus(DE3)-RIL cells</td><td>Strategene</td><td>#230245</td><td></td></tr><tr><td>Leupeptin</td><td>Roche Applied Science</td><td>#11034626001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche Applied Science</td><td>#11359053001</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Roche Applied Science</td><td>#10837091001</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>#DN25-1G</td><td></td></tr><tr><td>Isopropyl &amp;#946;-D-1-thiogalactopyranoside (IPTG)</td><td>Roche Applied Science</td><td>#11411446001</td><td></td></tr><tr><td>Amylose resin</td><td>New England Biolabs</td><td>#E8021L</td><td></td></tr><tr><td>Econo-Column chromatography column</td><td>BIO-RAD</td><td>#7372512</td><td></td></tr><tr><td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td><td>BIO-RAD</td><td>#732-4130</td><td></td></tr><tr><td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS15RH02</td><td></td></tr><tr><td>Superose 6 prep grade column</td><td>Amersham Bioscirnces</td><td>#17-0489-01</td><td></td></tr><tr><td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS0611</td><td></td></tr></tbody></table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

Die MGM101 Gen wurde vor 20 Jahren für seine Rolle bei der Aufrechterhaltung der mitochondrialen DNA identifiziert. Studien aus mehreren Gruppen haben vorgeschlagen, dass das Protein in der Mgm101 recombinational Reparatur der mitochondrialen DNA beteiligt ist. Neuere Untersuchungen haben gezeigt, dass Mgm101 der Rad52-Typ Rekombinationsprotein Familie verwandt ist. Diese Proteine ​​bilden große oligomere Ringe und fördern die Anlagerung homologen einzelne DNA-Moleküle. Allerdings hat die Charakterisierung von Mgm101 der Schwierigkeit bei der Herstellung des rekombinanten Proteins behindert. Hier wird ein zuverlässiges Verfahren zur Herstellung von rekombinantem Mgm101 beschrieben. Maltose Binding Protein (MBP)-markierten Mgm101 zunächst in Escherichia coli exprimiert. Das Fusionsprotein wird zunächst durch Amylose-Affinitätschromatographie gereinigt. Nach der Begrüßung durch proteolytische Spaltung freigesetzt wird Mgm101 von MBP durch Kationenaustauschchromatographieschritt getrennt. Monodispersen Mgm101 erhält danndurch Größenausschlusschromatographie. Eine Ausbeute von ~ 0,87 mg Mgm101 pro Liter Bakterienkultur kann routinemäßig erhalten. Das rekombinante Mgm101 eine minimale Verunreinigung der DNA. Die vorbereiteten Proben werden erfolgreich für biochemische, strukturelle und Single Particle-Image-Analysen von Mgm101 verwendet. Dieses Protokoll kann auch zur Herstellung von anderen großen oligomere DNA-bindende Proteine ​​falsch gefaltet und giftig für Bakterienzellen verwendet werden.

Mgm101 ist ein Rad52-bezogenen Rekombinationsprotein in Mitochondrien. Rad52 wurde ausgiebig für seine Rolle in der mitochondrialen DNA-Rekombination (Abbildung 1) untersucht. Rekombinante Mgm101 kann durch eine Drei-Schritt-Verfahren (Abbildung 2) hergestellt werden. Dies wird durch die Verwendung des MBP-Tagging Strategie Mgm101 in eine lösliche Form exprimiert werden und anschließend von dem Tag durch proteolytische Spaltung freigesetzt (Fig. 3) kann erleichtert. ...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Die Hefe-Mitochondrien nucleoid Protein, Mgm101, ist ein Rad52-Typ Rekombinationsprotein die große oligomere Ringe bildet. Ein Protokoll wird beschrieben, dass lösliches rekombinantes Mgm101 vorzubereiten mit dem Maltose Binding Protein (MBP)-Tagging-Strategie mit Kationenaustausch und Größenausschlusschromatographie gekoppelt.

Vorbereitung des Mgm101 Rekombinationsprotein von MBP-based Tagging-Strategie

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

preparation-mgm101-recombination-protein-mbp-based-tagging

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12.0K Aufrufe.  State University of New York Upstate Medical University. Die Hefe-Mitochondrien nucleoid Protein, Mgm101, ist ein Rad52-Typ Rekombinationsprotein die große oligomere Ringe bildet. Ein Protokoll wird beschrieben, dass lösliches rekombinantes Mgm101 vorzubereiten mit dem Maltose Binding Protein (MBP)-Tagging-Strategie mit Kationenaustausch und Größenausschlusschromatographie gekoppelt.

Serie: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Engineering künstliche Faktoren zur Speziellen Manipulation alternativen Spleißen in menschlichen Zellen

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Forschung

Genetik

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Induzierbares T7-RNA-Polymerase-vermitteltes Multigen-Expressionssystem, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-vermittelten gezielte Integration In Vivo mit einer Homologie-vermittelte Ende verbinden-basierte Strategie

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Genetische Manipulation in<em&gt; Δku80</em&gt; Die Stämme für Funktionelle Genomanalyse<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Immunologie und Infektion

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafisch-Modell der Neuroblastom-Metastasierung

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Krebsforschung

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Photoaktivierte Lokalisationsmikroskopie mit Bimolekulare Fluoreszenzkomplementation (BIFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Biotechnik

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Anreicherung nativer und rekombinanter extrazellulärer Vesikel von Mykobakterien

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subklonierung Plus-Insertion (SPI) - A Novel Recombineering Verfahren zur schnellen Bau der Gene Targeting Vektoren

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Biologie

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

Einstufige CRISPR-basierte Strategie zur endogenen Genmarkierung in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

Neurowissenschaften

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Vorbereitung des Mgm101 Rekombinationsprotein von MBP-based Tagging-Strategie

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