The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs

Abstract

The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

Keywords: drug selection; multiple transgenes; piggyBac; porcine embryonic fibroblasts; somatic cell nuclear transfer; transposon.

Figure 1

(A) Schematic representation of selectable marker expression vectors. Plasmid backbone is not shown in this figure. CAG, cytomegalovirus enhancer + chicken β-actin promoter; pA, poly(A) sites; hph, hygromycin phosphotransferase gene; PGKp, mouse phosphoglycerate kinase promoter; neo, neomycin resistance gene; pac, puromycin-N-acetyltransferase gene; bsr, blasticidin S deaminase gene; SV40p, SV40 early promoter; PB, acceptor site in piggyBac system; and Sh ble, a protein that binds to zeocin and prevents it from binding DNA; and (B) beneficial effects of piggyBac-based gene delivery for efficient acquisition of stable transfectants. PEFs were transfected with a single PB vector (pT-pac) in the presence or absence of a transposase expression vector, pTrans (pT-pac vs. pTrans + pT-pac in “single gene transfection”), as described in the Materials and Methods. Similarly, they were transfected with double PB vectors (pT-pac + pT-hph) in the presence or absence of pTrans (pT-pac + pT-hph vs. pTrans + pT-pac + pT-hph in “double gene transfection”). After drug selection, emerging colonies were counted by staining with Giemsa. Photographs taken after Giemsa staining are shown above each column, together with the number of colonies generated.

Figure 2

Acquisition of stable PEF transfectants after simultaneous transfection with seven PB vectors. (A) Fluorescence micrographs of PEFs one day after transfection in the presence (experimental group, Exp) or absence (control group, Cont) of pTrans. Note that in both transfection groups there are some cells exhibiting both green and red fluorescence, but no significant difference in gene transfer efficiency between these two groups was observed. Phase, taken under light; tdTomato, red fluorescence derived from tdTomato in pT-tdTomato; and EGFP, green fluorescence derived from EGFP in pT-EGFP. Scale bars = 20 µm; (B) fluorescence micrographs of stable PEF transfectants (mPB-1 and mPB-13). Note that mPB-1 exhibited both green and red fluorescence, whereas only green fluorescence was observed for mPB-13. The abbreviations above each figure are the same as shown in (A). Scale bars = 20 µm; (C) PCR analysis of stable PEF transfectants (numbered mPB-1 to 13). Genomic DNA isolated from each transfectant was subjected to regular PCR using the primer sets shown in the Materials and Methods. m, 100-bp ladder markers; lane C, normal PEFs; lane PC, control plasmids (pT-neo for detection of neo, pT-pac for detection of pac, pT-hph for detection of hph, pT-Sh ble for detection of Sh ble, pT-bsr for detection of bsr, pT-EGFP for detection of EGFP cDNA, and pT-tdTomato for detection of tdTomato cDNA); (D) determination of the number of copies of the introduced transposon DNA in the PEF transfectants (mPB-1 (lane 1), -2 (lane 2), and -3 (lane 3)). C1, C4, C7, and C10 indicate PEF DNA plus one, four, seven, or 10 copies of transposon DNA, respectively; (E) Assay of drug sensitivity in stable PEF transfectants. Cells (mPB-1, THEPN, and untransfected PEFs (PEF)) were plated in a 48-well plate, and cultured in medium containing G418, medium containing G418 + hygromycin B (hyg B) + puromycin (puro), or medium containing G418 + hyg B + puro + blasticidin S (bS) + zeocin (zeo), for 10 days. After culturing, cells were stained with Giemsa stain for visualization.

Figure 3

(A) Fluorescence micrographs of mMPEF-1 prior to SCNT. Note that there are some cells exhibiting both green and red fluorescence (arrowed), but other cells without any fluorescence (shown by arrowheads) in this clone. Phase, taken under light; tdTomato, red fluorescence derived from tdTomato in pT-tdTomato; and EGFP, green fluorescence derived from EGFP in pT-EGFP. Scale bars = 20 µm; (B) fluorescence micrographs of developing blastocysts derived from SCNT using mMPEF-1 as the SCNT donor. Phase, taken under light; tdTomato, red fluorescence derived from tdTomato in pT-tdTomato; and EGFP, green fluorescence derived from EGFP in pT-EGFP. Scale bars = 100 µm; and (C) PCR analysis of a single blastocyst derived from SCNT using mMPEF-1 as the SCNT donor. Genomic DNA isolated from each single blastocyst was subjected to WGA prior to PCR analysis. The data shown are the results from the nested PCR. M, 100-bp ladder marker; lanes 1 and 2, blastocysts showing both red and green fluorescence; lanes 3 and 4, blastocysts showing no fluorescence; lane C, normal PEFs; lane PC, control plasmids (pT-neo for detection of neo; pT-pac for detection of pac; pT-hph for detection of hph; pT-Sh ble for detection of Sh ble; pT-bsr for detection of bsr; pT-EGFP for detection of EGFP cDNA; pT-tdTomato for detection of tdTomato cDNA).

Figure 4

Future strategy to acquire porcine fibroblasts carrying multiple gene constructs using a PB-based gene delivery system. At least 5 plasmids (i.e., pT-AB, pT-CD, pT-EF, pT-GH and pT-IJ) can be used. In each plasmid, there is a unit conferring expression of specific drug resistance gene (i.e., neo) and a unit conferring expression of at least two types of proteins (i.e., A and B, C and D, E and F, G and H or I and J) under the control of upstream strong CAG-based promoter. Transfection of PEFs with these PB transposons and transposase expression vector pTrans and subsequent selection with multiple selective drugs will lead to generation of cells carrying multiple constructs in their genome.