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.
| Reid, Susan W; Tessarollo, Lino (2009) Isolation, microinjection and transfer of mouse blastocysts. Methods Mol Biol 530:269-85 |
| Tessarollo, Lino; Palko, Mary Ellen; Akagi, Keiko et al. (2009) Gene targeting in mouse embryonic stem cells. Methods Mol Biol 530:141-64 |
| Wang, Xin; Rao, Raghavendra Pralhada; Kosakowska-Cholody, Teresa et al. (2009) Mitochondrial degeneration and not apoptosis is the primary cause of embryonic lethality in ceramide transfer protein mutant mice. J Cell Biol 184:143-58 |
| Southon, Eileen; Tessarollo, Lino (2009) Manipulating mouse embryonic stem cells. Methods Mol Biol 530:165-85 |
| Wang, Lie; Wildt, Kathryn F; Zhu, Jinfang et al. (2008) Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells. Nat Immunol 9:1122-30 |
| Satyanarayana, Ande; Berthet, Cyril; Lopez-Molina, Javier et al. (2008) Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2. Development 135:3389-400 |
| Taylor, Gregory A; Rodriguiz, Ramona M; Greene, Robert I et al. (2008) Behavioral characterization of P311 knockout mice. Genes Brain Behav : |
| Zeng, Hongkui; Horie, Kyoji; Madisen, Linda et al. (2008) An inducible and reversible mouse genetic rescue system. PLoS Genet 4:e1000069 |
| Chiang, Y Jeffrey; Hsiao, Susan J; Yver, Dena et al. (2008) Tankyrase 1 and tankyrase 2 are essential but redundant for mouse embryonic development. PLoS One 3:e2639 |
