Using Modified Bacterial Artificial Chromosomes to Develop Transgenic Mouse Models Containing Human Genes with Disease Causing Mutations

Srilekha Kadimi

Document Type

Article

Publication Date

Summer 2023

Keywords

JMG

JAX Location

In: Student Reports, Summer 2023, The Jackson Laboratory

Abstract

The primary goal of this study was to develop a pipeline for BAC recombineering using a human gene containing mutations to make transgenesis a more targeted and efficient process to reduce the time required to generate new mouse models of human disease. The large insert capacity of bacterial artificial chromosomes (BACs) allows introduced transgenes to resist positional modifications and effectively build disease model systems to reflect pathogenesis. Using homologous recombination aided by a rpsL counterselection marker, construction of two donor DNAs for significant mutations on the TARDBP gene, a human gene associated with ALS, were attempted. Homologous recombination between the donor DNA and the BAC was unsuccessful and the process will continue to be optimized to allow the rpsL gene cassette to bind to the BAC genome rather than to the E. coli genome. A second aim of the study was to use BAC recombineering for subcloning into a low copy plasmid. This process had two goals. The first goal was to add attB sites to both sides of the gene of interest. The second goal was to increase the yield of the BAC. A minimal vector flanked by attB sites were successfully combined and transformed into E. coli. Homology arms surrounding the gene of interest have been attached to the construct via PCR, but with low concentration. Further work will be required to optimize this subcloning approach. This study has significant implications for making improved transgenic mouse models to model human disease and creating an efficient pipeline to be used in future studies.

Please contact the Joan Staats Library for information regarding this document.

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