PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

Abstract

We designed a new type of polyadenylation-signal (PAS) trap vector system in living mice, the piggyBac (PB) (PAS-trapping (EGFP)) gene trapping vector, which takes advantage of the efficient transposition ability of PB and efficient gene trap and insertional mutagenesis of PAS-trapping. The reporter gene of PB(PAS-trapping (EGFP)) is an EGFP gene with its own promoter, but lacking a poly(A) signal. Transgenic mouse lines carrying PB(PAS-trapping (EGFP)) and protamine 1 (Prm1) promoter-driven PB transposase transgenes (Prm1-PBase) were generated by microinjection. Male mice doubly positive for PB(PAS-trapping (EGFP)) and Prm1-PBase were crossed with WT females, generating offspring with various insertion mutations. We found that 44.8% (26/58) of pups were transposon-positive progenies. New transposon integrations comprised 26.9% (7/26) of the transposon-positive progenies. We found that 100% (5/5) of the EGFP fluorescence-positive mice had new trap insertions mediated by a PB transposon in transcriptional units. The direction of the EGFP gene in the vector was consistent with the direction of the endogenous gene reading frame. Furthermore, mice that were EGFP-PCR positive, but EGFP fluorescent negative, did not show successful gene trapping. Thus, the novel PB(PAS-trapping (EGFP)) system is an efficient genome-wide gene-trap mutagenesis in mice.

Mapping Viral Susceptibility Loci in Mice

“…In addition, various approaches have been used to increase the frequency of spontaneous mutations in classical inbred mouse lines. These approaches include treating male mice with mutagens such as N-ethyl-N-nitrosourea (ENU) (8), which leads to point mutations, and using transposons such as Sleeping Beauty (9) or piggyBac (10), which result in the insertion of specific DNA sequences randomly throughout the genome.…”

Identification of genes associated with cortical malformation using a transposon-mediated somatic mutagenesis screen in mice

“…In addition, the insertion sites in the genome are relatively easy to identify—compared to radiation and chemical mutagenesis. Thus PB is an effective way of forward genetic screens in a range of model organisms 19 – 21 .…”

Ablation of Zfhx4 results in early postnatal lethality by disrupting the respiratory center in mice

“…PiggyBac ( PB ) is a DNA transposon, and its insertion can ablate gene expression to facilitate the identification of gene function, making it a useful genetic manipulation tool in mice ( Ding et al, 2005 ; Li et al, 2016 ; Friedrich et al, 2019 ; Weber et al, 2019 ). To explore the biological function of Zfhx4 , we used a H362cR1 PB insertion mouse line, in which a modified PB transposon element was inserted into the third intron of the Zfhx4 gene ( Campbell et al, 2002 ; Ding et al, 2005 ; Sun et al, 2008 ).…”