우리는 다양한 장비를 사용할 수 있도록 유용 조언과 A. 와일드에 대한 E.의 고메스 감사하고 싶습니다. 이 작품은 건강 연구 (FRN 102725)에 대한 캐나다 연구소에서 AFP에 부여에 의해 지원되었다.

<ol>
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P., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+promotes+rapid+and+efficient+mRNA+export+in+mammalian+cells.">Splicing promotes rapid and efficient mRNA export in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3386-3391 (2008).</li><li>Palazzo, A. F., Eng, C. H., Schlaepfer, D. D., Marcantonio, E. E., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+stabilization+of+microtubules+by+integrin-+and+FAK-facilitated+Rho+signaling.">Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling.</a> Science. 303, 836-839 (2004).</li><li>Keller, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proper+alignment+of+the+microscope.">Proper alignment of the microscope.</a> Methods Cell Biol. 72, 45-55 (2003).</li><li>Mathews, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+chemical+modification+constraints+into+a+dynamic+programming+algorithm+for+prediction+of+RNA+secondary+structure.">Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 7287-7292 (2004).</li><li>G&#246;rlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal+sequence+receptor+has+a+second+subunit+and+is+part+of+a+translocation+complex+in+the+endoplasmic+reticulum+as+probed+by+bifunctional+reagents.">The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents.</a> J. Cell Biol. 111, 2283-2294 (1990).</li><li>Perales, R., Bentley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Cotranscriptionality":+the+transcription+elongation+complex+as+a+nexus+for+nuclear+transactions.">"Cotranscriptionality": the transcription elongation complex as a nexus for nuclear transactions.</a> Mol. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+copy+number+estimation+using+a+mouse+whole-genome+oligonucleotide+microarray.">Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray.</a> Genome Biol. 6, 61-61 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Microinjection 다른 세포 프로세스의 숫자를 연구하는 데 사용할 수있는 강력한 도구입니다. 기존의 세포 transfection 달리, microinjection은 연구자가 효과적으로 매우 좁은 기간 내에 세포 핵에 핵산을 소개하실 수 있습니다. 그 결과, 하나는 mRNA 수출, mRNA의 현지화 및 단백질 합성의 속도를 결정하는 등 운동 분석을 수행할 수 있습니다. 또한, 실험의 기간은 비교적 짧은이기 때문에, 하나는 pleiotropic 효과를 발생하지 않고 짧은 시간에 걸쳐 내에 독성 화합물로 세포를 노출하실 수 있습니다. 또한, 그러한 지배 부정적인 단백질로 억제 또는 stimulatory 화합물은 핵산과 함께 공동 주입 수 있습니다. 마지막으로, microinjections은 종종 세포에 독성이 서로 다른 세포 기능을 교란 수 있습니다 transfection의 시약에 대한 요구 사항을 피할 수 있습니다. 플라스미드 DNA를 주사 대 mRNA 삽입하는 핵산은 고려의 숫자에 따라 달라집니다 중 선택할 수 있습니다. 플라스미드 DNA를 주사를 따라 RNA 중합 효소 II는 mRNA를 합성뿐만 아니라, 초기 사본 12,13에 단백질 요소를 모집뿐만 아니라. 이러한 요소는 이러한 mRNA 처리, 원자력 수출 및 세포질 현지화 같은 이벤트를 제어 때문에, 플라스미드 DNA를 주사는 전사가 하류 프로세스에 결합하는 방법 파악하기 위해 사용될 수 있습니다. 전사가 핵 수출 14 결합 수 있지만, 우리는 주입 mRNA가 약간 빠른 생체내 베꼈는데 mRNA 6 이상의 수출 것으로 나타났습니다. 이에 대한 이유가 지금 분명 아니지만,이 결과로 새로 합성된 mRNA는 RNA 중합 효소 II에서 나온다 것을 제안할 수 있습니다, 그것은 그 저능​​아 핵 수출 단지로 묶여있을 수 있습니다. mRNA 주사 몇 가지 이점이 있습니다. 첫째, 연구자는 세포가 RNA의 전반적인 신진 대사를 조절하는 방법에 영향을 미칠 수있는, RNA 효소 억제제를 사용할 필요가 없습니다. 둘째, RNA는 사출하기 전에 체외에서 수정할 수 있습니다. 예를 들어, mRNAs 이러한 기능이 핵 수출 6 미치는 영향 평가하기 위해 다양한 5 &#39;모자 analogues 또는 폴리 (A) 꼬리 길이와 합성 수 있습니다. 셋째, 주입 RNA의 정확한 금액은 대략 예상하실 수 있습니다. 위에서 설명한 프로토콜에 따라, 우리는 전형적인 포유 동물 세포 (400,000에 850,000 분자) 15 성적의 총 개수에 비해 약 20,000 50,000 분자가 비교적 작고 각 핵에 주입하는 예상하고있다. mRNA의 주사와 함께 가장 큰 단점은 microinjection 동안 세포질로 주입 유체의 일부 누수가 불가 피한 때문입니다. 세포질에 직접 주입 mRNA가 오랜 기간 (A. 집이되지 않은 관찰)을 통해 안정되기 때문에 별도의주의가 세포를 계량하는 선택로 이동합니다. 일반적으로 우리는 OG - dextran의 분포에 의해 평가 핵에 주입 유체의&gt; 90 %를받은 세포를 분석할 수 있습니다.

<table><tbody><tr><td>Injection buffer</td><td>Sigma-Aldrich</td><td></td><td>140 mM KCl and 10 mM HEPES, pH 7.4</td></tr><tr><td>Oregon Green 488-70kD Dextran</td><td>Invitrogen</td><td>D7173</td><td>Stock of 10mg/ml in injection buffer can be stored for longs periods of time at -80&amp;#x00B0;C</td></tr><tr><td>Borosilicate capillary tubes</td><td>World Precision Instruments, Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Gel loading pipette tips for loading injection needles</td><td>Eppendorf</td><td>022351656</td><td></td></tr><tr><td>Microscope</td><td>Nikon Instruments</td><td>Eclipse Ti-S</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige International</td><td>NT-88-V3MSH</td><td></td></tr><tr><td>Syringe for injection</td><td>BD Biosciences</td><td>512311</td><td></td></tr><tr><td>PBS Buffer</td><td>Sigma-Aldrich</td><td></td><td>137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 2 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 7.4</td></tr><tr><td>SSC Buffer</td><td>Sigma-Aldrich</td><td></td><td>150 mM NaCl, 15 mM sodium citrate, pH 7.4</td></tr><tr><td>4% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15686</td><td>Stock of 37% Paraformaldehyde is diluted in PBS</td></tr><tr><td>0.1% Triton X-100 (Surfact-Amps)</td><td>Thermo Fisher Scientific, Inc.</td><td>28314</td><td>Stock of 10% Triton X-100 is diluted in PBS</td></tr><tr><td>Formamide</td><td>Fisher Scientific</td><td>F84-1</td><td></td></tr><tr><td>Dextran sulfate</td><td>Fisher Scientific</td><td>BP1585-100</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; tRNA</td><td>Sigma-Aldrich</td><td>R1753</td><td></td></tr><tr><td>Vanadyl riboside complex (VRC)</td><td>Sigma-Aldrich</td><td>R3380</td><td></td></tr><tr><td>Whatman filter paper</td><td>VWR international</td><td>28298-020</td><td></td></tr><tr><td>Vibration control table</td><td>Kinetic Systems</td><td>5702E-3036-21</td><td></td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>&amp;alpha;-amanitin</td><td>Sigma-Aldrich</td><td>A2263</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Vacuum Grease</td><td>Dow Corning</td><td>695400008</td><td></td></tr><tr><td>T7 RNA Polymerase</td><td>New England Biolabs</td><td>M0251S</td><td></td></tr><tr><td>Poly(A) Polymerase</td><td>Ambion</td><td>AM1350</td><td></td></tr><tr><td>Hybridization Buffer</td><td>Sigma-Aldrich</td><td></td><td>100mg/ml dextran sulfate, 5mM VRC, 150mM NaCl, 15mM sodium citrate, 0.5mg/ml &lt;em&gt;E. coli&lt;/em&gt; tRNA, 60% formamide</td></tr></tbody></table>

analysis of mrna nuclear export kinetics in mammalian cells by microinjection

eukaryotes에서 메신저 RNA (mRNA)가 핵에 베꼈는데되고 번역 기계에 액세스하는 세포질에 수출해야합니다. mRNA의 핵 수출이 Xenopus oocytes의 1과 같은 효모 2 Drosophila S2 파생된 셀 3 호선으로 취급하기 쉬운 유전자 생물에서 널리 연구되어 있지만, 몇 가지 연구는 포유 동물 세포에서 실시했다. 또한 포유 동물 체세포의 mRNA 수출의 반응 속도론은 간접적으로 4,5 유추 수 있습니다. 포유 동물 조직 배양 세포에서 mRNA의 핵 수출 속도론을 평가하기 위해, 우리는 현장의 하이브리드화 (물고기)의 형광와 결합 microinjection의 전원을 고용 분석을 개발했습니다. 이러한 assays는 포유 동물 세포에 mRNAs의 대부분이 splicing에 의존하게 6,7으로 수출, 또는 신호 순서 코딩 영역 (SSCR) 6과 같은 특정 RNA의 시퀀스를 필요로하게됩니다 입증하는 데 사용되었습니다. 이 분석에서는, 세포는 mRNA 합성 또는 관심있는 유전자를 포함하는 플라스미드 DNA와 중 체외에서 microinjected 있습니다. microinjected 세포 그런 다음 고정 및 RNA의 하위 세포 로컬 리 제이션이 물고기를 사용하여 평가됩니다 여러 시간 지점 incubated 수 있습니다. 전사는 핵산의 추가 후 몇 시간이 발생 transfection에 대조적으로, DNA 또는 mRNA의 microinjection은 빠르게 표현 가능하며 정확한 운동 데이터의 생성을 허용합니다.

Watch this Scientific Journal Video about Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection at JoVE.com

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

여기 형광와 결합 microinjection의 전원을 고용 분석을 설명<em&gt; 현장에서</em정확하게 포유류의 체세포에서 mRNA의 핵 수출 속도론을 측정하기 위해&gt; 하이브 리다이 제이션.

Microinjection하여 포유 동물 세포에서 mRNA 원자력 수출 속도론 분석

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

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Research

JoVE Journal

Biology

15.2K 조회수.  University of Toronto. 여기 형광와 결합 microinjection의 전원을 고용 분석을 설명 현장에서 하이브 리다이 제이션.

비디오: Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

형광 단백질의 발현에<em&gt; 창고 기</em&gt; 미 수정 난자에 mRNA의 주입에 의한

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

연구

발생학

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

감수 분열 동안 인간 유전자 기능을 평가 하기 위해 마우스 Oocytes를 사용 하 여 나

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

유전학

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus Laevis의 Oocytes의 Microinjection

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

생물학

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

포유류 세포에서 인트로닉 마이크로RNA 성숙을 분석하기 위한 리포터 분석

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

암 연구학

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Dissociated 성인 포유류의 뉴런으로 DNA의 Microinjection Intranuclear

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

모터 뉴런과 유사한 NSC-34 세포 내에서 실시간으로 핵 세포질 수송을 측정하는 분석

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

신경과학

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

RIP 칩을 사용하여 세포 추출물에서 mRNAs, microRNAs 및 Ribonucleoprotein 단지의 단백질 구성 요소의 분리 및 확인에 방법

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

미세 주입을 사용하여 인간 헬라 세포에서 스플리세오소말 snRNA 국소화 분석

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Microinjection하여 포유 동물 세포에서 mRNA 원자력 수출 속도론 분석

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