A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis

Zhao, Liang, Ng, Ee Ting and Koopman, Peter A. (2014) A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis. Developmental Dynamics, 243 9: 1086-1094. doi:10.1002/dvdy.24153

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Author Zhao, Liang
Ng, Ee Ting
Koopman, Peter A.
Title A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis
Formatted title
A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis
Journal name Developmental Dynamics   Check publisher's open access policy
ISSN 1058-8388
Publication date 2014-06-25
Year available 2014
Sub-type Article (original research)
DOI 10.1002/dvdy.24153
Open Access Status Not Open Access
Volume 243
Issue 9
Start page 1086
End page 1094
Total pages 9
Place of publication Hoboken, NJ, United States
Publisher John Wiley & Sons
Language eng
Formatted abstract
Background: Bacterial artificial chromosomes (BACs) have become increasingly popular vectors for making transgenic mice, as they are able to carry large genomic DNA fragments that in many cases are needed to reproduce the endogenous gene expression pattern. However, the efficiency of BAC transgenesis is generally low, and gene transfer to BAC vectors by recombination-mediated engineering (recombineering) is time-consuming and technically demanding.

Results and Conclusions: We present an enhanced system, comprising a BAC vector retrofitted with piggyBac DNA transposon elements and attL (Gateway) docking sites, that obviates these problems. Using this system, a gene-of-interest (such as a reporter gene) is transferred to the vector in a one-step in vitro reaction, and piggyBac transposition mediates transgene integration at high efficiency when microinjected into mouse zygotes with piggyBac transposase mRNA. We establish proof-of-principle for this system using a Wilms tumour-1 (Wt1) BAC to drive expression of an mCherry-2A-EGFP (RG) reporter gene, which yielded transgenic mice at a frequency of 33%, and recapitulated endogenous WT1 expression in developing gonads, kidneys and heart. The system we describe is applicable to any BAC transgenesis strategy.
Keyword Transgenic mice
Sex determination
Testis development
Kidney development
Heart development
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2015 Collection
Institute for Molecular Bioscience - Publications
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Citation counts: TR Web of Science Citation Count  Cited 4 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 4 times in Scopus Article | Citations
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Created: Wed, 02 Jul 2014, 19:48:23 EST by Susan Allen on behalf of Institute for Molecular Bioscience