In the past several years, our cores top priority has been keeping up with the latest developments in genome engineering methods, especially the CRIPSR (clustered regularly interspaced short palindromic repeat) technology. We can now efficiently generate knockout mice by creating small frame shift mutations or generating large deletions by cutting with two or more CRISPR constructs. We can also use single strand oligonucleotides as donors to efficiently knockin point mutations or insert small pieces of DNA, such as loxP or various tags. In the last 12 months, we have assisted more than twenty NIH laboratories in over three dozen knockout/knockin projects. We also completed four conditional knockout projects using the CRISPR method. We have also made progress in knocking in large genes using double strand donor vectors. 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 generating transgenic mice using the pronuclear microinjection method, generating knockout mice using the conventional ES cell-mediated homologous recombination, carrying out mouse in vitro fertilization, re-deriving and resurrecting mouse lines. We have also made technical advancements in using such traditional transgenic and ES cell-mediated methods. For examples, we have produced excellent chimeric mice using an ES cell line derived from the PWD mouse strain, and have inserted a large transgene into the Rosa26 locus using the so-called site-specific (TARGATT) transgenic technology. In addition, we are also continually expanding our capabilities in using immunocompromised mice to support stem cell-related research. Our services in this area have extended to beyond teratoma assays, i.e. searching for representative tissues from all three germ layers. The cell types we inject have broadened to include differentiated cells and cancer cells, and the assays have also expanded to analyzing human cell engraftment and testing the efficacy of experimental drugs.

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