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

View Article: PubMed Central - PubMed

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.

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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.
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ijms-17-01424-f002: 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.

Mentions: To demonstrate the utility of the PB-based gene delivery system for acquiring transfectants carrying multiple gene constructs, we transfected PEFs with a cocktail containing seven PB vectors (five drug resistant and two fluorescent plasmids) and pTrans (experimental group) or containing only seven PB vectors (control group). To assess transfection efficiency, fluorescence was inspected using a fluorescence microscope one day after transfection. No appreciable difference in the rate of cells with red and/or green fluorescence was noted (Exp vs. Cont in Figure 2A). However, the rate of stable colonies generated after drug selection was significantly different between the experimental and control groups. In the experimental group, there were 10–13 colonies, whereas no colonies were seen in the control group (Table 1). Inspection of fluorescence revealed that of 13 colonies tested, 12 had both red and green fluorescence (as exemplified by mPB-1 clone in Figure 2B), whereas the remaining colony expressed only green fluorescence (as exemplified by the mPB-13 clone in Figure 2B). These 13 colonies were subjected to colony isolation using paper methods as described in the Materials and Methods; all colonies were successfully propagated. PCR analysis of genomic DNA isolated from these colonies demonstrated that all colonies had five unique drug-resistance genes (Figure 2C). As expected, colonies (mPB-1 to mPB-12) with both red and green fluorescence had both EGFP and tdTomato cDNAs (Figure 2C). The mPB-13 colony exhibiting only green fluorescence had EGFP, but not tdTomato cDNA (Figure 2C).


The piggyBac -Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
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.
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ijms-17-01424-f002: 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.
Mentions: To demonstrate the utility of the PB-based gene delivery system for acquiring transfectants carrying multiple gene constructs, we transfected PEFs with a cocktail containing seven PB vectors (five drug resistant and two fluorescent plasmids) and pTrans (experimental group) or containing only seven PB vectors (control group). To assess transfection efficiency, fluorescence was inspected using a fluorescence microscope one day after transfection. No appreciable difference in the rate of cells with red and/or green fluorescence was noted (Exp vs. Cont in Figure 2A). However, the rate of stable colonies generated after drug selection was significantly different between the experimental and control groups. In the experimental group, there were 10–13 colonies, whereas no colonies were seen in the control group (Table 1). Inspection of fluorescence revealed that of 13 colonies tested, 12 had both red and green fluorescence (as exemplified by mPB-1 clone in Figure 2B), whereas the remaining colony expressed only green fluorescence (as exemplified by the mPB-13 clone in Figure 2B). These 13 colonies were subjected to colony isolation using paper methods as described in the Materials and Methods; all colonies were successfully propagated. PCR analysis of genomic DNA isolated from these colonies demonstrated that all colonies had five unique drug-resistance genes (Figure 2C). As expected, colonies (mPB-1 to mPB-12) with both red and green fluorescence had both EGFP and tdTomato cDNAs (Figure 2C). The mPB-13 colony exhibiting only green fluorescence had EGFP, but not tdTomato cDNA (Figure 2C).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus