转基因
转基因
整合酶
重组工程
生物
遗传学
重组酶
噬菌体
计算生物学
Cre重组酶
表情盒
DNA
基因靶向
转基因小鼠
同源重组
重组DNA
载体(分子生物学)
基因
重组
胚胎发生
生殖生物学
大肠杆菌
作者
Benjamin E. Low,Vishnu Hosur,Simon Lesbirel,Michael V. Wiles
出处
期刊:Research Square - Research Square
日期:2021-11-03
标识
DOI:10.21203/rs.3.rs-1008117/v1
摘要
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 ~43kb 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 combined with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.
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