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Integrase-Mediated Targeted Transgenics Through Pronuclear Microinjection

  • Ruby Yanru Chen-TsaiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2066)

Abstract

Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal and provides an extremely powerful tool to dissect the molecular mechanisms of disease. Transgenic mouse models made by microinjection of DNA into zygotic pronuclei, in particular, have been widely used by the genetics community for over 35 years. However, up till 5 years ago, it remained a rather crude approach: injected sequences randomly insert in multiple copies as concatemers, and they can be mutagenic and have variable, ectopic, or silenced expression depending on the site of integration, a phenomenon called position effects. As a result, multiple lines are required in order to confirm appropriate transgene expression. This can be partially overcome by flanking transgenes with insulator sequences to protect the transgene from influence of surrounding regulatory elements. Large (<300 kb) BAC-based transgenic vectors have also been shown to be more resistant to position effects. However, animals carrying extra copies of fairly large regions of the genome could have unpredictable phenotypes.

These problems can be overcome by targeting the transgene to a specific chromosomal locus via homologous recombination in embryonic stem (ES) cells. However, this method is significantly more laborious and time consuming, as it involves creation of modified ES cells and mouse chimeras, as well as eventual germline transmission of the transgene.

Here, I describe an integrase-based approach, trademarked as “TARGATT™” (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Key words

Pronuclear microinjection PhiC31 integrase TARGATT™ Site-specific transgenesis Rosa26 knockin H11 locus 

Notes

Acknowledgments

The author thanks all Applied StemCell, Inc. employees who have contributed to improving the TARGATT™ technologies. Special thanks to Maki Ogawa for helping with the figures.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Applied StemCell, Inc.MilpitasUSA

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