During the past several years, our core has devoted a large portion of our efforts on keeping up with the fast advancing genome engineering technologies. We have successfully used the ZFN (zinc finger nucleases), TALEN (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeat) methods to generate knockout/knockin cell lines and mouse models. In the last 12 months, we have significantly improved the efficiency and consistency for using the CRISPR method to generate knockout mouse models, as well as for using oligonucleotides-mediated homologous recombination to knockin point mutations or insert small pieces of DNA. We have successfully completed more than two dozen knockout or knockin projects using the CRISPR method. However, it still remains challenging for us to use the CRIPSR method to create conditional knockout mice in one step, and to use targeting vector-based homologous recombination to insert large pieces of DNA, such as reporter genes. We are currently attempting to solve these technical difficulties using various experimental approaches. Besides developing the new genome engineering technologies, our core is continuing to provide a variety of services using the classical mouse genetic and reproductive methodologies, including the generation of transgenic lines using the pronuclear microinjection method, the generation of knockout mice using the standard ES cell-mediated homologous recombination and blastocyst microinjection, re-deriving and resurrecting mouse lines. We have also imported the TARGATT mouse line, which enables the insertion of a single copy transgene into a predefined genomic locus. We have also been providing services on injecting stem cells into immunocompromised mice for testing their ability to form teratomas. In the past year, the cell types we injected have broadened to beyond iPSCs/ESCs, and the analysis has also extended to beyond searching for tissues from all three germ layers.
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