The past several years has been an exciting period for genome engineering, our core has spent a significant portion of our efforts on keeping up with the latest technological advancements, especially the CRIPSR (clustered regularly interspaced short palindromic repeat) technology. In the past 12 months, we have assisted over two dozen NIH laboratories in more than forty knockout/knockin mouse projects. Our efficiencies for using CRISPR to generate knockout mice and oligo-mediated small knockin mice are now very high. We can also efficiently generate large deletions by co-cutting with two or more guide RNAs. We have also made significant progress in knocking in large genes using double strand donor vectors. In the past year, we have also completed nearly one dozen conditional knockout and targeting vector-based large knockin mouse lines using the CRIPSR method. In collaboration with Dr. Lothar Hennighausen's laboratory, we have compiled a list of CRISPR-created on-target mutations in 632 founder mice at 17 independent loci (see Ref #3 in Bibliography section), which serves as a useful reference for the in vivo gene-editing field. We have also obtained a grant for conducting a comprehensive analysis of off-target mutations in some of these founder mice. In collaboration with Dr. Harry Malech's laboratory, we have published a paper on the inactivation of the Cybb gene in immunocompromised (NSG) mice (see Ref # 5 in Bibliography section), which has become a useful tool for studying infectious diseases. Of course, we are continuing to make transgenic and knockout mice using the classical pronuclear microinjection and ES cell-mediated homologous recombination methods, as well as using the so-called TARGATT technology to insert single copy transgenes into predefined safe harbor genomic loci. We have also been utilizing our expertise in stem cells, assisted reproductive technologies, and animal surgery to provide a range of other services, including re-deriving mice, resurrecting mouse lines using in vitro fertilization, assessing stem cell differentiation propensities through teratoma formation assay. In collaboration with the iPS core, Surgery core, Pathology core, and scientists at George Washington University, we are exploring methods for transplanting human iPSC-derived cardiomyocytes, either alone or embedded in 3-D printed scaffolds, into immunocompromised mice.

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Support Year
10
Fiscal Year
2017
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U.S. National Heart Lung and Blood Inst
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Lin, Yongshun; Liu, Huimin; Klein, Michael et al. (2018) Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs. Sci Rep 8:5907
Zhang, Yingfan; Liu, Chengyu; Adelstein, Robert S et al. (2018) Replacing nonmuscle myosin 2A with myosin 2C1 permits gastrulation but not placenta vascular development in mice. Mol Biol Cell 29:2326-2335
Jiao, Delong; Cai, Zhenyu; Choksi, Swati et al. (2018) Necroptosis of tumor cells leads to tumor necrosis and promotes tumor metastasis. Cell Res 28:868-870
Zhuang, Lenan; Jang, Younghoon; Park, Young-Kwon et al. (2018) Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function. Nat Commun 9:1796
Xing, Shaojun; Shao, Peng; Li, Fengyin et al. (2018) Tle corepressors are differentially partitioned to instruct CD8+ T cell lineage choice and identity. J Exp Med 215:2211-2226
Liu, Tanbin; Hu, Yi; Guo, Shiyin et al. (2018) Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells. PLoS One 13:e0192641
Vizcardo, Raul; Klemen, Nicholas D; Islam, S M Rafiqul et al. (2018) Generation of Tumor Antigen-Specific iPSC-Derived Thymic Emigrants Using a 3D Thymic Culture System. Cell Rep 22:3175-3190
Deis, Jessica A; Guo, Hong; Wu, Yingjie et al. (2018) Lipocalin 2 regulates retinoic acid-induced activation of beige adipocytes. J Mol Endocrinol :
Lee, Hye Kyung; Willi, Michaela; Wang, Chaochen et al. (2017) Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice. Nucleic Acids Res 45:4606-4618
Xu, Zhe; Xing, Shaojun; Shan, Qiang et al. (2017) Cutting Edge: ?-Catenin-Interacting Tcf1 Isoforms Are Essential for Thymocyte Survival but Dispensable for Thymic Maturation Transitions. J Immunol 198:3404-3409

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