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. 2009 Apr;19(4):667-73.
doi: 10.1101/gr.085621.108. Epub 2009 Feb 20.

A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells

Wei Wang  1 , Allan Bradley, Yue Huang
Affiliations

A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells

Wei Wang et al. Genome Res. 2009 Apr.

Abstract

Cultured mouse or human embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Chemical or insertional mutagenesis of Blm-deficient mouse ES cells can be used to generate genome-wide libraries of homozygous mutant ES cells, which are the substrates for conducting phenotype-driven loss-of-function genetic screens. However, the existing insertional mutation libraries are limited by incomplete genomic coverage. In this study, we have explored the use of piggyBac (PB) transposon-mediated mutagenesis to extend the genomic coverage of mutation libraries in Blm-deficient ES cells. A library composed of 14,000 individual gene-trap clones was generated and a recessive genetic screen conducted to identify cells with defects in DNA mismatch repair (MMR) genes. Independent mutations in all known genes of the pathway Msh2, Msh6, Pms2, and Mlh1 were recovered in these screens. The genomic coverage in this library confirms its utility as a new genetic resource for conducting recessive genetic screens in mammalian cells.

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Figures

Figure 1.
Figure 1.
PiggyBac revertible gene-trap vectors, PBGTV1 and PBGTV2. (A) The PBGTV1 series contain the minimal PB terminal DNA repeats and an adenovirus splice acceptor (SA) Beta-geo gene-trap cassette flanked by FRT sites. Three versions were constructed in which the coding sequence of Beta-geo has different reading frames. (B) The PBGTV2 series contain the minimal PB terminal DNA repeats and a loxP flanked gene-trap cassette consisting of an En2 splice acceptor (En2SA) T2A-Beta-gal-T2A-Neo cassette. Four versions were constructed. In vectors 0, 1, and 2, the coding sequence of T2A-Beta-gal-T2A-Neo has different reading frames. In vector K, an ATG start codon was used for the Beta-gal coding sequence. pA, bovine growth hormone polyA signal; SA, adenovirus major late gene splice acceptor; En2SA, mouse engrailed 2 gene splice acceptor; T2A peptide is a self-cleaving short peptide from Picornavirus (Szymczak et al. 2004).
Figure 2.
Figure 2.
Copy number analysis of PB insertions. (A) PB transposition in the presence of transposase. Five micrograms of transposon gene-trap vector (PBGTV1-1) were electorporated with or without 10 μg of transposase expressing plasmid into 5 × 106 AB2.2 cells. A representative picture of plates obtained from G418 selection. (B) Southern blot analysis of gene-trap clones reveals host/transposon junction fragment in each clone. RV, EcoRV. LacZ, probe, an 800-bp ClaI fragment from pSAgeo (Soriano et al. 1991). Black triangles indicate the endogenous neomycin locus in NGG5-3 ES cells. (C) Summary of copy numbers obtained using different transfection conditions at a single transposase concentration, 20 μg.
Figure 3.
Figure 3.
Strategy to generate and isolate 6-TG resistant clones. Blm−/−, Hprt1+/+ (NGG5-3) cells were used to generate gene-trap mutations by transposon gene-trap mutagenesis. The gene-trapped clones were selected with G418, pooled and expanded for 14 generations. After expansion, each pool, which consists of heterozygous and homozygous clones, was screened using 6-TG, and resistant clones were picked for further analysis. 6-Thioguanine (6TG) is utilized by the purine nucleotide synthesis pathway and is incorporated into replicating DNA leading to mismatchs within the DNA duplex. The MMR complex recognizes this type of mismatch and initiates multiple rounds of repair, which generates single- and double-strand DNA breaks. These activate signaling pathways and lead to cell cycle arrest and apoptosis. Thus, 6-TG is a sensitive selective agent for the isolation of DNA MMR mutants.
Figure 4.
Figure 4.
Analysis of 6-TGR clones. (A) Insertion sites of transposons in four DNA mismatch repair genes shown by arrows. The insertion–host junctions were identified by SpPCR and sequence analysis. The vector and reading frames are indicated. (B) Homozygosity analysis of five mutant clones; Msh2-m1, Pms2-m1, Mlh1-m1, Mlh1-m2, and Msh6-m1. Genomic PCR analysis was conducted with primers for the PB3′ and PB5′ transposon/host junctions (mutant alleles, marked as “3′” and “5′,” respectively) or with genomic primers which span the insertion site (wild-type alleles). The wild-type genomic fragments “+” are absent in gene-trap mutant clones but were amplified from Blm-deficient ES cells (marked as “wt”), which indicated that transposon insertions are homozygous in all cases except Mlh1-m2. (C) Wild-type and fusion transcripts in gene-trap mutants. RT-PCR was performed with primers for the upstream exon of the relevant mutated gene to lacZ gene (marked as “Msh2-Z, Pms2-Z, Mlh1-Z, and Msh6-Z”) as well as for the wild-type alleles. The wild-type transcripts are detected in nonmutated Blm-deficient control samples (marked as “wt”) but were absent in all four mutant clones. Fusion transcripts of the expected size were detected in three mutant clones, Msh2-m1, Pms2-m1, and Mlh1-m1.
Figure 5.
Figure 5.
Reversibility of Mlh1-m1 mutation with PBase. (A) Low-density survival assay demonstrating 6-TG sensitivity of three revertant clones. (B) Genomic PCR analysis of 96 reversal candidates with primers for the PB3′ IR and sequences upstream of insertion site of Mlh1 (marked as “3′”) detected the transposon/host junction of the expected size. Representative candidate clones including three reverted ones are shown. The genomic PCR product fragments (marked as “+”) are absent in two mutant clones (C2 and C4) but were amplified from three revertants (C1, C3, and C5). (C) Expression of wild-type Mlh1 and fusion Mlh1-lacZ transcripts in gene-trap mutants. RT-PCR with primers for exon2 of Mlh1 and lacZ (marked as “M-Z”) detected fusion transcripts of the expected size from all 96 clones. The wild-type transcripts (marked as “Mlh1”) are absent in clones C2 and C4 but are found in the three revertants (C1, C3, and C5). (D) Southern blot analysis of Mlh1-m1 revertant clones identifies a new integration of the transposon in revertant clone 5. EV, EcoRV. Probe, LacZ.
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