Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer

Abstract

The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

Fig.1. Generation of transgenic porcine fetal fibroblast colonies by piggyBac transposon-mediated gene transfer

Structure of plasmid vectors, donor pB CMV-neo-EGFP (A) and helper mPBase (B); pB 5’TR, pB transposon 5’ terminal repeat element. pB 3’TR, pB transposon 3’ terminal repeat element. CMV, CMV promoter. neo, neomycin-resistant gene. EGFP, enhanced green fluorescence protein gene. Probe, the probe used for Southern blot. bGH polyA, bovine growth hormone gene polyA. (C). Surviving fetal fibroblast colonies in 10 cm dishes after transfection with linearized pEGFP-N1 or co-transfection with donor vector pB CMV-neo-EGFP and helper vector mPBase, and 2 week selection with G418. The pictures of the dishes with G418-resistant cell colonies were taken under blue light. (D). Colony count of G418-resistant cells in the dishes. The experiment was performed in triplicate and the data is presented as mean ± SD. A mean of 10 and 309 colonies was observed in cultures transfected with linearized plasmid pEGFP-N1, or co-transfected with donor vector pB CMV-neo-EGFP and helper vector mPBase, respectively.

Fig.2. EGFP expression in transgenic pigs produced by pB transposon-mediated gene transfer

(A). A representative picture showing green florescence was not visualized on wild-type (WT) piglets, but was clearly observed on transgenic (TG) piglets, especially on their noses and hooves (indicated by the arrows) under blue light. (B). A representative picture showing green florescence was also clearly observed on the organs of TG piglets but not on that of WT piglets under blue light.

Fig.3. Genetic analyses of transgenic pigs and FF clones produced by pB transposon-mediated gene transfer

(A). PCR analysis of EGFP, pB 5’-TRE, pB 3’-TRE and pB transposase expression gene cassette in the genome of transgenic pigs. A 609 bp fragment of EGFP gene was amplified from the genomic DNA of all the transgenic pigs but not from wild-type pigs. PCR products for pB 5’-TRE, pB 3’-TRE and pB transposase were not detected. P, positive control using plasmid DNA of donor vector pB CMV-neo-EGFP as template for amplification of EGFP gene, or using plasmid DNA of helper vector mPBase as template for amplification of pB transposase gene expression cassette. (B). PCR analysis of EGFP, pB 5’-TRE, pB 3’-TRE and pB transposase expression gene cassette in the genome of eight individual FF clones. Again we detected EGFP, but no PCR products for pB 5’-TRE, pB 3’-TRE and pB transposase. (C). Southern blot analysis of EGFP in transgenic pigs. 1x, 2x and 3x, represents plasmid positive control respectively harboring 1 copy, 2 copies and 3 copies of transgene per pig genome. Southern blot analysis of positive control was carried out with a mixture of 10 μg of wild-type pigs’ genomic DNA and 2.57×10^-5 μg (1 copy), or 5.14×10^-5 μg (2 copies), or 7.71×10^-5 μg (3 copies) of donor vector pB CMV-neo-EGFP plasmid DNA as total starting DNA. The amount of plasmid DNA used to mix with wild-type pigs’ genomic DNA for Southern blot analysis of positive controls was calculated based on the following equation: copy number (1, or 2, or 3) × plasmid length (6.94×10³ bp) / pig genome whole length (27×10⁸ bp) = amount of plasmid DNA / amount of WT pig genomic DNA (10 μg). Six bands (indicated by white arrows) were detected from the genome of transgenic pigs, and the intensity of each band was similar with that of 1 copy positive control, suggesting the transgene was integrated in at least 6 sites of each transgenic pig genome, and that each insertion site contains only 1 copy of transgene. (D). Southern blot analysis of EGFP in ten individual transgenic and two wild-type FF clones. (E). Real-time quantitative PCR analysis of transgene copy number per transgenic pig genome. Data is presented as mean ± SD (n=4).

Fig.4. Identification of pB transposon integration sites in transgenic pig’s genome

Chromosomal DNA sequences flanking the inserted pB transposon were identified by sequencing the inverse PCR products. The obtained flanking DNA sequences were then used to blast against Sus scrofa (pig) genomic DNA database to find out their location in pig genome. All identified insertion site sequences contain the TTAA sequences which is indicative of the insertion border between the pB transposon and flanking genomic DNA. (A). An identified integration site of pB transposon on chromosome 4. The pB transposon was inserted in between 716 bp and 717 bp of GenBank sequence (GenBank number NW_003534687.2, length=657315 bp). (B). An identified integration site of pB transposon on chromosome 2. The pB transposon was inserted in between 96558 bp and 96559 bp of GenBank sequence (GenBank number NW_003299559.3, length=172486 bp).