Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons

Abstract

The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.

Fig. 1.

DNA transposon vectors and stable transfection rates into a chicken cell line. (A) Diagram of the piggyBac and Tol2 vectors used in this study. Tol2-CGIP and PB-CGIP contain identical reporter cassettes between the ITRs. The CAG enhancer/promoter drives expression of a GFP-IRES–puromycin reporter construct. Flanking insulator elements (Ins) from the chicken β-globin locus were inserted adjacent to the ITRs in the piggyBac construct, PB-CGIP+2I. (B) DF-1 cells were transfected with transposon constructs with (+) or without (−) transposase and assayed 3 wk after transfection for GFP expression by flow cytometry. Data represent three independent experiments. The stable transfection efficiencies are corrected for the initial rate of transfection. Error bars, SEM. *P < 0.05.

Fig. 2.

Stable genetic modification of primordial germ cells using piggyBac and Tol2 vectors. Chicken PGCs were transfected with transposon constructs with (+) or without (−) transposase and visualized a minimum of 2 wk after transfection for GFP expression. (A) Minus transposase control; (B) PB-CGIP vector; (C) Tol2-CGIP vector. (Scale bar, 50 μm.) (D) Comparison of stable transfection rates of piggyBac and Tol2 transposons in PGCs. Data are corrected for transfection efficiency and represent a minimum of four independent experiments, using two lines of PGCs. Error bars, SEM. **P < 0.01.

Fig. 3.

Analysis of the affect of increasing the amount of transposase on stable transfection and genomic integration events in primordial germ cells. (A) Chicken PGCs were transfected with 1.0 μg of a PB-CGIP or a Tol2-CGIP vector and increasing amounts (micrograms) of the appropriate DNA transposase and visualized a minimum of 2 wk after transfection for GFP expression. All transfection contained equal total amounts of plasmid DNA. The data are from four independent experiments. The stable transfection efficiencies are corrected for the initial rate of transfection. (B) Genomic DNA from pools of stably transfected PGCs was analyzed for presence and copy number of the piggyBac transposon by digestion with BamHI to generate junction fragments and hybridized with a probe to the GFP sequence. Arrowheads, junctional fragments.

Fig. 4.

Distribution of genomic integration sites of the Tol2 transposon in primordial germ cells. The integration sites for 50 Tol2-CGIP integrations were identified using inverse PCR and the transposon–genomic junction fragments were mapped to the chromosomes of the chicken genome.

Fig. 5.

Gene trap of endogenous transcripts in primordial germ cells. (A) A minimal Tol2 transposable element containing a β-globin splice acceptor (SA) followed by a SA-GFP–puromycin reporter gene containing a self-cleaving peptide sequence and a poly(A) sequence. (B) GFP and brightfield images of a representative PGC culture that was transfected with the gene-trap vector and the transposase expression plasmid and selected with puromycin. (Scale bar, 100 μm.) (C) Fusion transcripts identified by RACE PCR analysis. Amino acids coding sequences are indicated in boldface type and the initiation codon for GFP is highlighted. The splice donor sites of the trapped exons and the gene-trap acceptor sites are underlined. The endogenous exon sequences fused to the transposon sequence are shown in italics. KCKB, ENSGALT00000018766; NMT1, ENSGALT00000001256. (D) A schematic representation of transposon integration sites in the trapped loci and the primers used to verify the fusion transcripts. (E) Independent PCRs were performed on cDNA isolated from the gene-trap PGCs with the indicated primers to verify the fusion transcripts. A primer located in the puromycin gene did not produce a PCR product, suggesting that a truncated transcript was produced.

Fig. 6.

Primordial germ cells modified with transposon vectors are germ-line competent. (A) A piggyBac vector containing a CAGmyrGFP-IRES-Puro reporter gene was transfected into PGCs and selected with puromycin. (Scale bar, 50 μm.) (B) Cross-section of a day 3 embryo, 1 d after injection of GFP-expressing PGCs, contained GFP⁺ cells in the genital ridge. Arrow, left genital ridge. (C and D) GFP⁺ cells in the gonads in day 5 (C) and day 10 (D) chicken embryos. (E) Cross-section of seminiferous tubules of an adult cockerel hatched after injection with GFP-expressing PGCs. GFP⁺ cells are located adjacent to the basement membrane of the tubules. [Scale bars (B–E), 100 μm.] (F) G₁ offspring of the adult cockerel injected with GFP-expressing PGCs and visualized for GFP expression. A nontransgenic sibling is on the right. (G) A schematic representation of the piggyBac insertion site in the first intron of the PARD6B gene of the G₁ offspring (ENSGALT00000012978). Red, piggyBac ITRs; boldface type, genomic DNA.

Fig. P1.

The genetic modification of chicken primordial germ cells using DNA transposons. 1. PGCs are isolated from chicken embryonic blood and propagated in vitro. 2. DNA transposons are introduced into PGCs and stably integrate into the genome. GOI, gene of interest. 3. The modified PGCs are injected into host embryos and raised to sexual maturity. 4. The genetically modified PGCs form functional gametes and produce offspring containing the DNA transposon.