NEI Genetic Engineering Core

National Eye Institute

Investigators

Linked publications & trials

Abstract

Summary Despite the ongoing pandemic of Covid-19, we accomplished a total of 26 genetic engineering projects either through CRISPR-mediated mutagenesis in vivo or classic homologous recombination in mouse embryonic stem cells. Some are highly interesting and are summarized below: 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. A de novo dominant neomorphic missense variant (c.926C>G:p.S309W) in SP7, a transcription factor critical for osteoblast maturation and bone formation, was observed in a patient with craniosynostosis, cranial hyperostosis, and long bone fragility. The same mutation was introduced into the mouse genome by CRISPR-mediated homologous recombination using an oligo carrying the targeted conversion. Mice with the corresponding variant showed a complex skeletal phenotype like those observed in the human patient. The mutation altered the binding specificity of SP7 from AT-rich motifs to a GC-consensus sequence (typical of other SP family members) and produced an aberrant gene expression profile, including increased expression of Col1a1 and endogenous Sp7, but decreased expression of genes involved in matrix mineralization. The study identifies a novel pathogenic mechanism in which a mutation in a transcription factor shifts DNA binding specificity. Age related macular degeneration (AMD) in humans, manifested as vision loss at older age, has been a subject of intense investigation. New candidate genes have been recently discovered by Dr. Anand Swaroop's lab at NEI, and GEC continued engineering additional mutant alleles in mouse to provide genetic confirmation of AMD candidate genes derived from GWAS. These include loss-of-function alleles of Hcls1 and Mrpp3 genes. Additional allelic series of missense mutations targeting the mouse Tbc1d24 gene have also been constructed at GEC. Pathogenic variants of the Tbc1d24 gene lead to syndromic and non-syndromic hearing loss conditions in human. Similar disease models developed at GEC this year include additional Sp7 and Spin4 missense mutations for bone density disorders. 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; several knockin lines with reporters have also been developed. One of such examples is the development of a GFP knockin allele at the mouse GSX gene, a transcription factor critical in the development of retinal pigmented Epithelium (RPE) during embryogenesis, followed by the deletion mutations removing upstream enhancer elements to define the functions of each individual enhancers in regulating temporal and special pattern of GSX expression during eye morphogenesis. This project is still ongoing and will likely yield critical information regarding gene expression regulation on GSX gene. 3) Maintenance of genetically altered rodent strains: a) Genotyping operation: GEC genotyping services aided total of 15 laboratories most of which are in NEI intramural program. * isolated DNA from 13,165 mouse tail biopsy samples, almost doubled from last year's number * performed 6,611 PCR reactions to genotype mice in the facility, also doubled last year's work load. * acted on behalf of several laboratories sending mouse tissue samples to Transnetyx for genotyping services. b) Cryopreservation and rederivation operations: GEC Cryopreservation services assisted many laboratories from NEI in cryopreserving 38 lines, rederivation of 1 line, and rescuing of 3 lines.