Mouse genomic editing projects at GEC this year covered the following scientific areas: 1) Human disease modeling: It is often desirable to have mutations of human genetic conditions replicated in mouse so that the diseases can be modeled. In the last year, several such models were developed at GEC in collaboration with investigators at NEI, NIDCD and NICHD. Mutations in the Kcnj13 locus lead to autosomal-dominant snowflake vitreoretinal degeneration in human. An allelic series of 3 missense mutations in mouse Kcnj13 gene have been developed to model this human retina degeneration condition in mouse in collaboration with Dr. Fielding Hejtmanciks laboratory at NEI. Another allelic series of three missense mutations targeting the mouse Pnpla2 gene have also been constructed at GEC. Pathogenic variants of the Pnpla2 gene lead to human Oliver-McFarlane and Laurence-Moon syndromes manifested as trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Similar disease models include TBC1D24 and Clpp1 missense mutations for congenital hearing loss, and a Sp7 missense mutation for bone density disorders. Age related macular degeneration (AMD) in human, which is often manifested as vision loss at older age, has been a subject of intense investigation. Four new candidate genes have been recently discovered by Dr. Anand Swaroops lab at NEI, and GEC is now engineering the equivalent mutant alleles in mouse to provide genetic confirmation of the AMD candidate genes derived from GWAS. 2) Functional genomic studies of genes predicted to be important in physiology and pathology: Most of the current genome editing projects are aimed at simply understanding the functions of various genes relevant to NEI, as well as other participating IC research programs. Work in this area currently includes the construction of multiple loss-of-function mutations such as genes involved in the visual cascade and in survival of cones and rods. During the past year, we have worked on 50 different gene targeting projects at various stages. In order to achieve mutagenesis goals of all the projects above, we have conducted the following experimental procedures: (1) Genetic engineering: * Provided 76 consultations for genome editing and other projects to investigators from NEI and many other labs NIH-wide. * Designed and constructed 45 recombinant DNA clones through Gibson assembly for gene targeting in ES cells, CRISPR/Cas9-mediated gene knockin in mouse zygotes, and lentiviral construction. * Produced and characterized 242 guide RNAs by in vitro transcription assays for CRISPR-mediated genome editing. * performed 21 Surveyor assays for 16 chromosomal loci in pre-injection screening * performed 26 in vitro Cas9-guide RNA mediated DNA double strand DNA break assays to evaluate efficiency of guide RNA-Cas9 interactions prior to injection *Designed and performed 5097 genotyping assays by PCR to identify F0 founders of genome edited mice * Made 18 endotoxin-free, large scale DNA preparations of DNA constructs. * Conducted over 29 transfection assays on 10 gene targeting projects in ES cells, picked 4704 ES clones, expanded and cryopreserved over 4000 individual ES clones, expanded 29 positive ES clones and in addition, conducted at least 4512 genomic DNA extractions and the same number of genomic PCR reactions for screening of targeted mutations. * Prepared 70 CRISPR samples for electroporation, and 218 samples for injection. Improved methodology of CRISPR reagent preparation for microinjection to reduce RNA precipitation and increase success rate for injection. * Optimized CRISPR-mediated genome editing in mouse zygotes with CRISPR recombinant protein based RNP methodology. * Fine-tuned our novel approach to introduce knockin alleles with CRISPR technology, such as reporter alleles or epitope tags, in R1 ES cells without the need for a selection cassette. (A manuscript describing this method has been accepted.) * Provided training to several graduate students and postdoctoral fellows from NEI and other institutes over the subjects of genome editing and mouse ES cell technology. * Participated in teaching of 3 workshops on CRISPR technology. * Collaborated with 2 transgenic core facilities on campus and outside NIH on various genome editing projects. * microinjected 13 ES cell lines into mouse embryos to generate 42 chimeric mice. * microinjected 39 CRISPR/Cas9 constructs into fertilized mouse oocytes for 42 injection sessions to produce 52 mutant mice (16 knockin and 40 knockout) out of 208 F0 founder mice. *Successfully adapted and subsequently optimized the technology using electroporation as an efficient mean delivering genome modifying reagents into mouse zygotes. * Electroporated 33 CRISPR/Cas9 constructs (18 knock in projects and 15 deletion or indel projects) into fertilized mouse oocytes for 70 EP sessions to produce 111 mutant mice (12 knock/in and 99 indel or deletion out of 264 F0 founders that already genotyped), with a total of 370 F0 founder mice born (the remaining 106 are still waiting for genotyping data). * Performed 60 oviduct transfers to assist electroporation operations. *Tested GONAD electroporation, an in vivo approach introducing genome editing reagents into mouse zygotes. * Performed 110 weekly tests by either microinjection or electroporation (EP) of CRISPR reagents for most projects including 82 EP sessions and 28 microinjection sessions followed by 3787 (3168 embryos in EP testing and 619 for microinjection testing) in vitro culture experiments going from one cell embryos to blastocysts, and 1608 (1395 blastocyst for EP testing and 213 for microinjection testing) single embryo genomic PCR assays. (2) Genotyping operation: * isolated DNA from 13,801 mouse tail biopsy samples * performed 9,103 PCR reactions to genotype mice in the facility * provided 13,801 purified mouse genomic DNA samples to 21 investigators at NEI (3) Mouse colony management operation: * set up 1,103 matings to propagate mouse lines * completed or oversaw weaning, tagging, and tail biopsy of 14,752 mice born in the facility * made 654 mouse deliveries/transfers to researchers' labs/shipments * provided 198 consultations to researchers on breeding strategies/project planning (4) Cryopreservation and rederivation operations: * rederived 12 mouse lines * worked on cryopreservation of 54 mouse lines freezing 3140 mouse embryos at the two cell stage, and 380 straws of sperm * cryopreserved 2 lines of zebrafish as frozen sperm and validated 2 lines. Cryopreserved testes from one lines of zebrafish * performed assisted reproduction to save 16 mouse lines from extinction and/or reconstitute mouse lines from frozen stock * validated 12 lines of frozen mouse germplasm (sperm and embryos) These services and collaborative services were performed for 20 PIs from 5 NEI labs/branches (LI, LRCMB, N-NRL, OGVFB, OSD), plus 8 PIs from 4 other NIH institutes (NINDS, NICHD, NIDCD and NIAMS) and 1 investigator at Johns Hopkins University.
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