Genetically modified mice by means of homologous recombination are generated by injection of manipulated ES cells into recipient blastocysts. The injected blastocysts, following re-introduction into recipient foster mothers will produce chimeric mice in which the manipulated ES clones populate the germ line and transmit the desired mutation to the offspring. The technology to generate genetically modified chimeras involves 3 main sequential steps. 1- Engineering of the targeting vector to introduce the desired mutation into the mouse genome;2- Introduction of the targeting vector into mouse embryonic stem cells (ES cells) to accomplish homologous recombination;3- Injection with the targeted ES cells and immediate transfer of blastocysts into pseudo-pregnant recipient mothers. We provide diversified support to the CCR-NCI scientific community with counseling and technical help for all 3 different stages depending on the experience and needs of the investigator. 1-Engineering of the targeting vector. The generation of a targeting vector for homologous recombination in ES cells requires careful planning. It is of paramount importance for the overall success of a specific project that this step is well thought and planned. We provide scientific input for the designing of an optimal targeting vector. We make available to the investigators the best molecular tools to engineer the targeting vector including protocols and reagents for the use of the recombineering technology. Recombineering is a powerful tool that allows the generation of the desired DNA vectors in a relatively short period of time (Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet. 2001, 769-79). 2- Introduction of the targeting vector into the ES cells to accomplish homologous recombination. The targeting vector is introduced into mouse ES cells by electroporation. ES clones are positively selected for the presence of specific antibiotic resistance (for example neomycin, but also puromycin or blastycidin) and negatively by the presence of the Thymidine Kinase (TK) or Diphteria Toxin (DT) genes. Selected clones are then grown in duplicate and one set is given to the investigators for analysis of specific homologous recombination. 3- Injection of the targeted ES cells into mouse blastocysts and subsequent transfer into pseudo-pregnant recipient females. ES clones identified as correctly targeted are grown and expanded for the micro-injection into blastocysts at 3.5 days of gestation. The microinjected blastocysts are implanted into pseudo-pregnant recipient females who will generate chimeras derived from the blastocyst and the targeted ES clone. Coat color is used to score and identify the chimeras that will likely transmit the desired mutation to the progeny.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICBC011265-01
Application #
8158377
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2010
Total Cost
$890,554
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Xiong, Yulan; Neifert, Stewart; Karuppagounder, Senthilkumar S et al. (2017) Overexpression of Parkinson's Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and ?-Synuclein Pathology. eNeuro 4:
Tu, Zhaowei; Bayazit, Mustafa Bilal; Liu, Hongbin et al. (2017) Speedy A-Cdk2 binding mediates initial telomere-nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation. Proc Natl Acad Sci U S A 114:592-597
Hartford, Suzanne A; Chittela, Rajanikant; Ding, Xia et al. (2016) Interaction with PALB2 Is Essential for Maintenance of Genomic Integrity by BRCA2. PLoS Genet 12:e1006236
Tobe, Ryuta; Carlson, Bradley A; Huh, Jang Hoe et al. (2016) Selenophosphate synthetase 1 is an essential protein with roles in regulation of redox homoeostasis in mammals. Biochem J 473:2141-54
Chalamalasetty, Ravindra B; Ajima, Rieko; Garriock, Robert J et al. (2016) A new gain-of-function mouse line to study the role of Wnt3a in development and disease. Genesis 54:497-502
Zhao, Rong; Grunke, Stacy D; Keralapurath, Madhusudhanan M et al. (2016) Impaired Recall of Positional Memory following Chemogenetic Disruption of Place Field Stability. Cell Rep 16:793-804
Schliehe, Christopher; Flynn, Elizabeth K; Vilagos, Bojan et al. (2015) The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection. Nat Immunol 16:67-74
Anderson, Matthew J; Southon, Eileen; Tessarollo, Lino et al. (2015) Fgf3-Fgf4-cis: A new mouse line for studying Fgf functions during mouse development. Genesis :
Mahdi, Yassin; Xu, Xue-Ming; Carlson, Bradley A et al. (2015) Expression of Selenoproteins Is Maintained in Mice Carrying Mutations in SECp43, the tRNA Selenocysteine 1 Associated Protein (Trnau1ap). PLoS One 10:e0127349
Ajima, Rieko; Bisson, Joseph A; Helt, Jay-Christian et al. (2015) DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly. Dev Biol 408:126-39

Showing the most recent 10 out of 39 publications