Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 192 (4), 1235-48

Insertional Mutagenesis by a Hybrid piggyBac and Sleeping Beauty Transposon in the Rat

Affiliations

Insertional Mutagenesis by a Hybrid piggyBac and Sleeping Beauty Transposon in the Rat

Kenryo Furushima et al. Genetics.

Erratum in

  • Genetics. 2014 Nov;198(3):1345

Abstract

A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

Figures

Figure 1
Figure 1
Transposons for transgenic rat production. (A) Bhr2 and (B) Bhr7 transposon constructs. Bhr2 and Bhr7 contain terminal inverted repeat sequences recognized by piggyBac transposase (light gray arrowheads) and the inverted/direct repeat sequences recognized by Sleeping Beauty transposase (dark gray arrowheads). Pink and green circles are attB and attP sequences, respectively, not exploited in this study. The primers for initial iPCR are I1, I2, and IS2 (black arrowheads) and for nested iPCR are N1, N2, and NS2 (blue arrowheads). The primer for RT–PCR for Bhr2 is B2-RT (red arrowhead). The primers, Tyr-F and Tyr-R (green arrowheads), are used for detecting the transposon in the rat genome. A portion of the Tyr reporter gene in Bhr2 and the splice donor and a subregion of the 3′ IRES sequence in Bhr7 were used as probes for Southern blot analysis. Primer sequences for iPCR, genotyping, and RT–PCR are described in Table 1. SA, Ad2 splice acceptor; pA in Bhr2, SV40 polyadenylation signal; pA in Bhr7, human growth hormone polyadenylation signal; Pro, Tyr promoter; pA after Tyr, Tyr polyadenylation signal; IRES, internal ribosomal entry site; rtTA, reverse tetracycline transactivator; SD, splice donor of rabbit β-globin intron; A, ApaI; AL, ApaLI; B, BamHI; E, EcoRI; P, PvuII; and X, XbaI.
Figure 2
Figure 2
Generation of transgenic rats by piggyBac transposition. (A) Litter generated from piggyBac transposon Bhr2 injected to albino Sprague-Dawley rat zygotes. Bhr2 piggyBac transposon founder transgenic rats are pigmented. Sprague-Dawley rats are homozygous for the hooded mutation that restricts pigmentation to the head and dorsum. (B) Offspring from a piggyBac transposon founder male crossed with a Sprague-Dawley female. All progeny were pigmented and each individual had a different coat color. (C) Southern blot analysis using a Bhr7-specific probe, showing multiple transposon integrations in a founder (F) and their segregation in progeny (1–8) generated by crossing with a Sprague-Dawley female.
Figure 3
Figure 3
Segregation and analysis of initial piggyBac transposon insertions. (A) Southern blot analysis, showing the isolation of single Bhr7 transposon gene insertions in separate pedigrees. L, LOC685774; P, Phex; +, wild type. Bhr2 transposon insertion in the Gabrb1 locus (G). The Tyr probe used for Bhr2 Southern analysis recognizes a single band of ∼7.4 kb for the endogenous Tyr locus. The Bhr2 transposon insertion in the Gabrb1 locus shows a single band of ∼7.6 kb and therefore appears the same as wild type. (B) Coat color differences in rats with different single-transposon insertions. The rats with insertions in LOC685774 or Phex loci remained albino. (C) Rats with the Bhr2 transposon in the Gabrb1 locus. Tn/Tn rats are darker than Tn/+ rats. (D) Rats with the Bhr7 transposon in the Zbtb20 locus. Tn/Tn rats are darker than Tn/+ rats. (E) PCR genotyping of progeny from a cross between Gabrb1Bhr2 hemizygotes.
Figure 4
Figure 4
Diagram of transposon insertions in the rat genome. Yellow circles, initial transposon insertions; blue circles, insertions mobilized from Gabrb1Bhr2 or LOC685774Bhr7 by piggyBac transposase; red circles, insertions mobilized from Gabrb1Bhr2 or LOC685774Bhr7 by Sleeping Beauty transposase.
Figure 5
Figure 5
Generation and functional analysis of piggyBac and Sleeping Beauty transposase-expressing transgenic rat lines. (A) Diagram of UBC-SB and UBC-PB gene constructs. The Sleeping Beauty or piggyBac transposase expression is driven by the human UBC promoter and flanked by chicken hypersensitive site 4 transcriptional insulators (cHS4) to reduce the influence of chromosomal integration sites. Transposase transgenic rats were identified by PCR using UBC-F and SB-R or PB-R primers (black arrowheads). Primer sequences for genotyping are described in Table 1. pA, rabbit β-globin intron and polyadenylation signal. (B) Male containing the Bhr2 transposon in the Gabrb1 locus (left) and seed male containing the Bhr2 transposon in the Gabrb1 locus and Sleeping Beauty (center) or piggyBac (right) transposase transgenes. All seed rats exhibited coat color mosaicism. (C) Male containing the Bhr7 transposon in LOC685774 locus (left) and seed male containing the Bhr7 transposon in the LOC685774 locus and Sleeping Beauty transposase (center) or piggyBac transposase (right) transgene. All seed rats were pigmented.
Figure 6
Figure 6
Structure of the Bhr2 transposon insertion in the Gabrb1 locus and germline mobilization. (A) Diagram of the Bhr2 transposon insertion site in the Gabrb1 locus. Brown arrow shows the direction of Tyr transcription. Primers for genotyping, arrowheads. Gab-F and Gab-R can detect the wild-type allele and Gab-F and B2-3 can detect the transposon allele. Primers for RT–PCR analysis, red arrowheads; Ex, exon. (B) Coat colors of the progeny with new transposon insertions originating from the Gabrb1 Tn locus generated by crosses of seed males and transposase-expressing rats. Rats 1–3 have different pigmentation compared to the original Bhr2 transposon Gabrb1 rat G, Gabrb1 Tn/+ rat. (C) PCR analysis of genomic DNA. The Sleeping Beauty transposase transgene is no longer present in the progeny (1–3) carrying the transposon at a new position (top). The rat with the Bhr2 transposon in the Gabrb1 locus has a wild-type (450 bp) and transposon (628 bp) allele but the rats with the new transposition events only have the wild-type band, demonstrating that the Bhr2 transposon has transposed away from the Gabrb1 locus (bottom). Rats 1–3 and G are the same as in B. PC, positive control; Wt, wild type. (D) Transposition events revealed by Southern blot analysis of offspring from seed males using Tyr probe. The Bhr2 transposon in the Gabrb1 locus and the endogenous Tyr locus digested with PvuII resulted in 7.6 kb (G) and 7.4 kb (Wt) bands, respectively. Progeny generated from seed male with different coat colors compared to Gabrb1Bhr rats have an additional band (*). Progeny 2 likely has a new insertion that yields a band of similar size to the Gabrb1 insertion. Progeny 1–3 and G are the same as in C. (E) Mutagenesis strategy using Bhr2 transposon insertion in the Gabrb1 locus. The seed male carrying both the transposon and transposase transgene that has coat color mosaicism is bred with an albino female. Progeny carrying the transposon at a different locus should have a different coat color compared to the original Bhr2 Gabrb1 insertion. Primer sequences for genotyping and RT–PCR are described in Table 1.
Figure 7
Figure 7
Structure of the Bhr7 transposon insertion in the LOC685774 locus and germline remobilization. (A) Diagram of Bhr7 transposon insertion site in the LOC685774 locus. Brown arrow shows the direction of Tyr transcription. Primers for genotyping, arrowheads. LOC-F and LOC-R can detect the wild-type allele and LOC-F and PB-IR-5.2 can detect the transposon allele. Ex, exon. (B) Coat color of progeny 1 and 2 with new transposition insertion sites. (C) PCR analysis of genomic DNA for the piggyBac transposase transgene (top) and transposon insertion in LOC685774 (bottom). Top shows that the piggyBac transposase transgene is not present in the rats carrying the Bhr7 transposon at a new position (1 and 2). Bottom shows the transposon in the LOC685774 locus is not present in the pigmented progeny (1 and 2). Progeny 1 and 2 are the same as in B. L, Bhr7 transposon in the LOC685774 locus of a Tn/+ female; Wt, wild type, S, seed rat. (D) Transposition events revealed by Southern blot analysis of offspring from seed males using a Bhr7-specific probe. The seed male (S) has multiple bands because of multiple transposition events in somatic tissues. The progeny that are pigmented have single bands of different size compared to the Bhr7 transposon in the LOC685774 (1 and 2). 1, 2, L, Wt, and S are the same as in C. (E) Mutagenesis strategy using Bhr7 transposon insertion in the LOC685774 locus. Rats carrying the Bhr7 transposon in the X chromosome-linked LOC685774 locus are albino. The seed male rat carrying both the transposon and transposase transgene that shows coat color mosaicism is bred with an albino female. Progeny carrying the transposon at a different locus can become pigmented. Primer sequences for genotyping are described in Table 1.
Figure 8
Figure 8
Expression analysis in Gabrb1 mutant rats. Adult brain mRNA of wild-type (left) and Gabrb1 (Tn/Tn) (right) rats. Primers for RT–PCR (red arrowheads) are shown in Figures 1A and 6A. Primers G-RT5F/G-RT5R, G-RTIF/G-RTIR, and G-RT3F/G-RT3R amplify mRNA before (5), between (I), and after (3) the transposon insertion site, respectively. Primers G-RTIF/B2-RT amplify mRNA trapped by the transposon in the presence of reverse transcriptase (+RT). All primer sets do not amplify DNA in the absence of RT (−RT). The Tn/Tn rat shows trapped mRNA (Tn) and wild-type mRNA (I and 3). Primers b-act F/b-act R were used to detect β-actin expression (C). Primer sequences for RT–PCR are described in Table 1. m, 100-bp ladder DNA marker.

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback