Bxb1-Integrase and Modified Bacterial Artificial Chromosomes as a Tool for Site-Specific Transgenesis of Large Donor DNA into Mouse Models

Thomas Mehaffey

Document Type

Article

Publication Date

Summer 2022

Keywords

JMG

JAX Location

In: Student Reports, Summer 2022, The Jackson Laboratory

Abstract

The primary goal of this study is to further optimize the Bxb1-Integrase (Int) method of transgenesis to improve the versatility and reduce the time required to generate new mouse models of human disease. Working directly in mouse zygotes, this platform has previously succeeded in generating transgenic mice with donor DNA sequences up to 43 kb in length (Low et al., 2022). This study presents BAC recombineering as a novel method for introducing Bxb1- Int attachment sites into donor DNA, enabling the generation of even larger transgenes. Previously, standard cloning techniques using restriction enzymes or CRISPR/Cas9-mediated homology directed repair (HDR) were used to generate the donor plasmids for site integration. As a proof of concept for recombineering techniques and to create relevant new human disease models, construction of two DNA donors were attempted: (1) a human DMPK gene (~20 kb) and (2) a human PEX1 gene (~60 kb). Using recombineering, we succeeded in positioning one of the Bxb1-Int attB sites 5’ of the human PEX1 gene situated inside of the BAC. However, the recombineering efficiency was low (1/136 for PEX1 and 0/136 for DMPK) and so further optimization will be necessary to improve the selection and efficiency. A second aim of this study was to assess the function of the Bxb1 Int protein in vivo by direct delivery of protein into a HEK cell line carrying a reporter plasmid. While activity was confirmed when cells were transfected with a Bxb1 Int expressing plasmid, no activity was detected in cells targeted with protein, possibly due to no/low uptake of the protein by the cells. Further work will be required to fully evaluate this platform’s potential for Bxb1 Int protein assessment.

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