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          Institute: MPI für molekulare Zellbiologie und Genetik     Collection: MPI-CBG Publications 2013 (arch)     Display Documents



ID: 688573.0, MPI für molekulare Zellbiologie und Genetik / MPI-CBG Publications 2013 (arch)
Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes.
Authors:Rostovskaya, Mariya; Naumann, Ronald; Fu, Jun; Obst, Mandy; Mueller, Doris; Stewart, A Francis; Anastassiadis, Konstantinos
Date of Publication (YYYY-MM-DD):2013
Title of Journal:Genesis
Volume:51
Issue / Number:2
Start Page:135
End Page:141
Copyright:not available
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that theBACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems. genesis 51:135-141, 2013. © 2012 Wiley Periodicals, Inc.
External Publication Status:published
Document Type:Article
Communicated by:Lib
Affiliations:MPI für molekulare Zellbiologie und Genetik
Identifiers:LOCALID:5213
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