Document Type

Article

Publication Date

3-31-2022

Publication Title

Sci Rep

Keywords

JMG, Animals, Bacteriophages, COVID-19, DNA, Gene Transfer Techniques, Genetic Background, Integrases, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Inbred NOD, RNA, Viral, SARS-CoV-2

JAX Source

Sci Rep 2022 Mar 31; 12(1):5424

Volume

12

Issue

1

First Page

5424

Last Page

5424

ISSN

2045-2322

PMID

35361849

DOI

https://doi.org/10.1038/s41598-022-09445-w

Grant

CA034196, OD027052, OD021325, OD026635

Abstract

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Comments

We thank Cindy Avery and Todd Nason for excellent maintenance of mouse colonies, Richard S. Maser for building the Krt18-ACE2 construct, Peter Kutny and group at Genetic Engineering Technologies for MIJs. We also thank various groups who assisted us, especially the Reinholdt laboratory (Dr David Bergstrom and Tiffany Leidy-Davis at JAX), and the Steve Murray laboratory (Dr. Kathy Snow at JAX) for providing constructs. We are grateful to the Genome Technologies (GT) group at JAX for Sanger and Nanopore sequencing services. We thank Cindy Avery for her assistance in animal maintenance, Melissa Berry for her careful reading of the manuscript and assistance in generating the mouse strain nomenclature.

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