We would like to thank the Wellcome Trust Sanger Institute for the gifts of piggyBac transposon and piggyBac transposase. This work was supported by the Butcher grant of Colorado and the NIH R01 AG041801 (W.S.C).

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G., 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-throughput+splinkerette-PCR+method+for+the+isolation+and+sequencing+of+retroviral+insertion+sites.">A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.</a> Nat Protoc. 4, 789-798 (2009).</li><li>Wang, W., Bradley, A., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+piggyBac+transposon-based+genome-wide+library+of+insertionally+mutated+Blm-deficient+murine+ES+cells.">A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells.</a> Genome Res. 19, 667-673 (2009).</li><li>Chick, W. 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=Screening+for+stress-resistance+mutations+in+the+mouse.">Screening for stress-resistance mutations in the mouse.</a> Front Genet. 5, 310 (2014).</li><li>Salmon, A. B., 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=Fibroblast+cell+lines+from+young+adult+mice+of+long-lived+mutant+strains+are+resistant+to+multiple+forms+of+stress.">Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress.</a> Am J Physiol Endocrinol Metab. 289, 23-29 (2005).</li><li>Skarnes, W. 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The authors have nothing to disclose.

Forward genetic analysis allows for an unbiased interrogation of the genome for genes responsible for a specific phenotype. This method is very powerful to uncover novel gene functions. It has been widely used in lower organisms but not in mammal, such as the mouse, mainly due to the extremely high cost associated with the infrastructure and logistics that would entail. Here, we moved the genetic screening process to the ES cell culture platform, greatly increasing the efficiency and throughput in generating mutants a...

장수 스트레스 저항과 친밀한 관계를 가지고있다. 일반적으로, 수명이 긴 종들은 과산화수소, 파라콰트 (PQ), UV, 열, 중금속 1,2- 여러 스트레스쪽으로 저항을 증가 보여준다. 대조적으로, 스트레스 증가 감도 짧아 수명 및 / 또는 더 발생하기 쉬운 질환 표현형을 예측하는 경향이있다. 항산화 소기 경로가 긴 동물에게 스트레스 저항성을 부여하는데 중요한 역할을하는 것으로 추측하고있다. 그러나, 거의 예외없이, 다양한 항산화 제 효소 (예를 들면, SOD)의 조작과 형질 전환 동물의 다양한 연구가 산화제 소거 효소의 수준을 증가 시키면 수명 또는 건강 스팬 3을 증가시키지 않음을 나타낸다. 이러한 데이터는 지속적으로 수명이 긴 동물에서 관찰 된 스트레스 저항 특성이 아직 발견되는 다른 세포 경로에 의해 매개되는 것이 좋습니다. 우리는 편견 전달했다방법은 돌연변이 유전자에 배양 배아 줄기 (ES) 세포에서 스트레스 내성 표현형을 부여 할 수있는 유전자를 동정을 행 하였다. ES 세포는이 연구에서 두 가지 장점을 제공한다 : (1) 유전자 정교한 조작이 ES 세포의 게놈을 수정은 가능하다; 및 (2) 스크린으로부터 회수 스트레스 내성 ES 세포는 직접 수명 및 건강 범위를 측정하기 위해 전체 동물 연구에 신속한 변환을 허용 마우스 생산에 사용될 수있다. 이보고에서는, 대립 유전자가 BLM 테트라 사이클린 반응성 소자에 의해 제어 된 C9에 ES 세포주의 사용을 설명했다. 독시 싸이클린 (D​​OX)의 치료는 일시적으로 자매 염색 분체 교환의 증가 사고로 이어지는 BLM의 발현을 해제. 이 단기적으로는 BLM 노크 아웃 이질 집단 내에서 동형 접합 돌연변이의 생성을 허용 스트레스 저항 열성 돌연변이가 심사 과정에서 캡처 할 수 있도록. 우리또한 무작위 게놈에서 유전자 돌연변이 폴리 트랩 카세트 (PB-UPA)를 삽입 돌연변이로 피기 백 (PB) 트랜스포존의 사용을 설명했다. 폴리 트랩에 의해 유전자의 파괴와 세포 G418 내성이되었고, 유전자 트랩 돌연변이 (유전자 트랩 라이브러리)의 컬렉션을 만들고, 이후에 강한 스트레스했다 돌연변이 클론 상영 될 수 있도록 복구 할 수 있습니다. 선택으로부터 회수 스트레스 내성 클론 삽입의 수 (qPCR에)의 점에서, 분자의 기술에 의해 다소 빠르게 특성화 될 수 삽입 사이트 (splinkerette PCR), 파쇄 유전자 (BLAST)의 신원, 및 그 발현 량 ( RT-qPCR에). PB 삽입은 야생형 DNA 서열을 복원하고, 따라서 응력의 저항 손실을 테스트 할 클론의 MPB 트랜스의 일시적 발현으로 remobilized 될 수있다. 이들은 이전에 수행해야 돌연변이의 인과 관계를 확인하는 강력한 방법이다비싼 마우스 생산. 이전의 연구는 스트레스에 노출 된 세포가 만능의 4,5을 잃은 것으로 나타났다. 따라서,이 프로토콜에서, 스트레스로 처리되지 않는다 돌연변이 세포의 집합 복제, 보존 성공적인 마우스 생산에 중요하다. 우리 연구소는 모두 요청에 따라 다른 연구자가 사용할 수있는, C9 ES 세포 라인과 PB-UPA 벡터를 설계하고있다. 여기에보고 된 프로토콜은 스트레스 내성 클론 (그림 1B)를 분리하기 위해 복제 도금과 스트레스의 선택에 따라 PB-UPA (그림 1A)와 유전자 갇혀 ES 세포의 드 노보 라이브러리의 생성에 의해 시작됩니다. 우리는 파라콰트, 세포 내부의 강력한 활성 산소 발생기 선택을 보여 주었다. 사실상, 예를 들어 임의의 세포 독성 화합물 또는 독소, ER 스트레스 요인 (예, 및 쌥시 가르 긴 tunicamycin), 신경 세포의 산화제 (예를 들어, MPP +, 6- 히드 록시 도파민 및 로테 논), 열, 또는 무거운금속 (예를 들면, CD, SE는) 각각 내성 돌연변이 체에 대해 선택하는 방법에 적용 할 수있다.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Vector&lt;/strong&gt;</td></tr><tr><td>PB-UPA</td><td></td><td></td><td></td></tr><tr><td>mPBase</td><td></td><td></td><td></td></tr><tr><td>mPBase&lt;sup&gt;Puro&lt;/sup&gt;</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td></tr><tr><td>500-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>250-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU02RE</td><td></td></tr><tr><td>50-ml Steriflip-GV filters</td><td>EMD Millipore</td><td>SE1M179M6</td><td></td></tr><tr><td>KO DMEM</td><td>Life Technologies</td><td>10829-018</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>FBS</td><td>Tissue Culture Biologicals</td><td>104</td><td></td></tr><tr><td>Heat Inactivated FBS</td><td>Sigma-Aldrich</td><td>F4135-500</td><td></td></tr><tr><td>LIF</td><td>EMD Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140148</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Methyl Viologen dichloride (Paraquat)</td><td>Sigma-Aldrich</td><td>856177</td><td></td></tr><tr><td>Dimethyl Sulphoxide Hybri-MAX</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>EmbryoMAX 0.1% gelatin</td><td>EMD Millipore</td><td>ES006B</td><td></td></tr><tr><td>DPBS/Modified</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>T25 Flask</td><td>Corning</td><td>353108</td><td></td></tr><tr><td>T75 flask</td><td>Corning</td><td>353135</td><td></td></tr><tr><td>100-mm&amp;nbsp; plate</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>150-mm&amp;nbsp; plate</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>96-well&amp;nbsp; plate</td><td>Corning</td><td>3585</td><td></td></tr><tr><td>96-well U-bottom plate</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>50-ml reservoir&amp;nbsp;</td><td>Corning</td><td>4870</td><td></td></tr><tr><td>15-ml tubes</td><td>VWR International, LLC</td><td>82050-276</td><td></td></tr><tr><td>Primary Mouse Embryonic Fibroblasts</td><td>EMD Millipore</td><td>PMEF-NL</td><td></td></tr><tr><td>DR4 Mouse Embryonic Fibroblasts</td><td>Applied StemCell</td><td>ASF-1001</td><td></td></tr><tr><td>Mitomycin C</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP25312</td><td></td></tr><tr><td>Geneticin (G418)</td><td>Life Technologies</td><td>11811-023</td><td></td></tr><tr><td>Doxycycline</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP26531</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td>377267</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5702</td><td></td></tr><tr><td>TC10 cell counter</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counting Slides (for TC10)</td><td>Bio-Rad</td><td>1450011</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electroporation&lt;/strong&gt;</td></tr><tr><td>Gene Pulser Xcell</td><td>Bio-Rad</td><td>1652611</td><td></td></tr><tr><td>Gene Pulser Cuvettes (4&amp;#160;mm gap)</td><td>Bio-Rad</td><td>1652088</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Molecular Biology&lt;/strong&gt;</td></tr><tr><td>Thermal Cycler</td><td>Eppendorf</td><td>Mastercylcer ep Gradient&amp;nbsp; S</td><td></td></tr><tr><td>Puregene Core kit B</td><td>Qiagen</td><td>158745</td><td></td></tr><tr><td>Topo-TA Cloning kit</td><td>Life Technologies&amp;nbsp;</td><td>450030</td><td></td></tr><tr><td>High Capacity cDNA synthesis kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>NaCl</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP358-212</td><td></td></tr><tr><td>100% ethanol</td><td>Decon Laboratories, Inc.</td><td>2716</td><td></td></tr><tr><td>Double Processed Tissue Culture Water</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>Sau3A1</td><td>New England BioLabs</td><td>R0169L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202T</td><td></td></tr><tr><td>EcoRV</td><td>New England BioLabs</td><td>R3195S</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;96-well Lysis Buffer (Ramires-Solis &lt;em&gt;et al.&lt;/em&gt; 1992)&lt;/strong&gt;</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher BioReagents&amp;nbsp;</td><td>A144-212</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L5777</td><td></td></tr><tr><td>Proteinase-K</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1700</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electrophoresis&lt;/strong&gt;</td></tr><tr><td>Mini-Sub Cell GT</td><td>Bio-Rad</td><td>170-4469EDU</td><td></td></tr><tr><td>LE Agarose&amp;nbsp;</td><td>GeneMate</td><td>E3120500</td><td></td></tr><tr><td>Ethidium Bromide&amp;nbsp;</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1302</td><td></td></tr><tr><td>100 BP DNA Ladder</td><td>New England BioLabs</td><td>N3231S</td><td></td></tr><tr><td>1 Kb DNA Ladder</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr><tr><td>2-log DNA Ladder</td><td>New England BioLabs</td><td>N3200L</td><td></td></tr></tbody></table>

forward genetic approach to uncover stress resistance genes in mice &#8212; a high-throughput screen in es cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

전형적인 돌연변이 유발 실험에서는 트랜 PB-UPA 및 MPB의 트랜스와 3 × 10 7 ES 세포의 전체로 구성되어있다. 두 독립적 인 실험에서 생성 된 유전자 포획 ES 세포의 숫자는 표 1에 요약되어있다. 유전자의 포집 효율은 약 0.04 %이다. 결합 된 유전자 트랩 라이브러리는 22,400 독립적 인 돌연변이 마우스 게놈에 대한 전체 범위 (23,000 코딩 유전자)를 포함한다. 과도한 따르면 반복 돌연...

Watch this Scientific Journal Video about Forward Genetic Approach to Uncover Stress Resistance Genes in Mice Ã¢â‚¬â€ A High-throughput Screen in ES Cells at JoVE.com

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

스트레스 저항은 장수의 특징 중 하나이며 유 전적으로 규율하는 것으로 알려져있다. 여기, 우리는 장수 연구에 마우스 모델을 개발하기 위해있는 배아 줄기 세포에 스트레스 저항을 부여 돌연변이 선별 편견 높은 처리량 방법을 개발했다.

앞으로 유전 접근은 마우스의 스트레스 저항성 유전자를 밝히기 위해서 - ES 세포에서 높은 처리량 화면

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

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JoVE Journal

Developmental Biology

8.1K 조회수.  University of Colorado Denver.  스트레스 저항은 장수의 특징 중 하나이며 유 전적으로 규율하는 것으로 알려져있다. 여기, 우리는 장수 연구에 마우스 모델을 개발하기 위해있는 배아 줄기 세포에 스트레스 저항을 부여 돌연변이 선별 편견 높은 처리량 방법을 개발했다. 

비디오: Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

마우스 배아 줄기 세포 사용 하 여 남부 럽 연쇄 반응에서에서 동종 재결합 이벤트의 식별

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

연구

유전학

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

재조합 효소 매개 카세트 교환을 사용하여 마우스 배아 줄기 세포의 구조 - 기능 연구

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

발생학

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

유전자 변형 마우스의 고효율 생산을 위한 동결 해동 배아 사용

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

정자의 대체로 마우스 파르테노유전학 Haploid 배아 줄기 세포의 응용 프로그램

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

유전자 조작 마우스 모델 개발을 위한 배아 줄기 세포에서 CRISPR/Cas9 매개 고효율 유전자 표적화

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

마우스 배아 줄기 세포 유지 보수 및 자체 갱신을 조절 유전자를 공부하는 Oct4GiP 리포터 분석

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

생물학

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

S. cerevisiae 에 단백질 기반 상속에 대 한 높은 처리량 검열

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans의 생리적 스트레스 반응 측정

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Schizosaccharomyces pombe에서 다중 복제 억제 인자의 식별을위한 유전자 스크리닝

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).