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Ryu, J.* ; Chan, W.* ; Wettengel, J.M. ; Hanna, C.B.* ; Burwitz, B.J.* ; Hennebold, J.D.* ; Bimber, B.N.*

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR.

Mol.Ther.-Methods Clin. Dev. 24, 241-254 (2022)

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Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Ggene Editing ; Integration Site Mapping ; Lentiviral Transduction ; Piggybac Transposase ; Transgenic Embryos

ISSN (print) / ISBN 2329-0501

e-ISSN 2329-0501

Journal Molecular Therapy. Methods & Clinical Development

Quellenangaben Volume: 24, Pages: 241-254

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Virology (VIRO)