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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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(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.
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ijms-17-01424-f001: (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.

Mentions: We transfected PEFs with single or double PB vectors with pTrans, a PB transposase expression vector (Figure 1A), to test gene transfer efficiency in the PB-based gene delivery system. As controls, PB vectors (without pTrans) were concomitantly introduced as described in the Materials and Methods. The gene transfer efficiency was evaluated by calculating the number of emerging stable transfectants after drug selection. The results are shown in Figure 1B. As expected, transfection with a single PB vector (pT-pac) + pTrans yielded 176 colonies, but with pT-pac alone resulted in only 45 colonies, indicating approximately four-fold higher gene transfer efficiency in this PB-based system. Transfection with double PB vectors (pT-pac + pT-hph) + pTrans yielded 22 colonies, whereas the double PB vectors alone failed to generate any viable colonies. Thus, gene transfer efficiency in transfectants with double PB vectors was reduced approximately seven-fold compared to that observed in transfectants with only a single PB vector. Given these results, we concluded that the PB system confers higher gene transfer efficiency in porcine cells.


The piggyBac -Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
(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.
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Related In: Results  -  Collection

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ijms-17-01424-f001: (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.
Mentions: We transfected PEFs with single or double PB vectors with pTrans, a PB transposase expression vector (Figure 1A), to test gene transfer efficiency in the PB-based gene delivery system. As controls, PB vectors (without pTrans) were concomitantly introduced as described in the Materials and Methods. The gene transfer efficiency was evaluated by calculating the number of emerging stable transfectants after drug selection. The results are shown in Figure 1B. As expected, transfection with a single PB vector (pT-pac) + pTrans yielded 176 colonies, but with pT-pac alone resulted in only 45 colonies, indicating approximately four-fold higher gene transfer efficiency in this PB-based system. Transfection with double PB vectors (pT-pac + pT-hph) + pTrans yielded 22 colonies, whereas the double PB vectors alone failed to generate any viable colonies. Thus, gene transfer efficiency in transfectants with double PB vectors was reduced approximately seven-fold compared to that observed in transfectants with only a single PB vector. Given these results, we concluded that the PB system confers higher gene transfer efficiency in porcine cells.

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