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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.

No MeSH data available.


(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).
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ijms-17-01424-f003: (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).

Mentions: To demonstrate that the nuclei carrying multiple constructs prepared using the PB-based gene delivery system have the potential to develop as SCNT-treated embryos, microminipig-derived PEF clones termed as mMPEF were used as the nuclear donors. In a preliminary test, we found that nuclei from the transfected PEFs derived from a Clawn miniature pig as well as its intact parental cells were unable to promote development of SCNT embryos, probably due to their reduced developmental potential, which might have occurred during cell cultivation (data not shown). We, therefore, decided to use mMPEF as SCNT donors. We obtained a candidate clone (termed mMPEF-1) from microminipig-derived PEFs after transfection with seven PB vectors (five drug resistant and two fluorescent plasmids) and pTrans and subsequent selection with drugs. The microphotograph of mMPEF-1 is shown in Figure 3A. This clone had cells showing both red and green fluorescence and those with no fluorescence in a ratio of approximately 3:2. The latter cells may have all the drug resistance genes, but lack both fluorescent genes. SCNT using mMPEF-1 revealed that 18.1% (4/22) of the SCNT-treated embryos developed to normal blastocysts. As expected, 50% (2/4) of these embryos expressed both fluorescent markers (Figure 3B), but the other embryos did not fluoresce (data not shown). To confirm this event at the molecular biological level, they were singly isolated, lysed, and subjected to WGA. PCR analysis of these WGA samples demonstrated that blastocysts expressing both fluorescent markers had seven PB vectors (lanes one and two in Figure 3C), whereas those expressing no fluorescence lacked both fluorescent genes, but had five drug resistance genes (lanes three and four in Figure 3C). These results suggest that PB-mediated simultaneous integration of multiple constructs into the genome of porcine cells does not affect the development of cloned embryos.


The piggyBac -Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
(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).
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ijms-17-01424-f003: (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).
Mentions: To demonstrate that the nuclei carrying multiple constructs prepared using the PB-based gene delivery system have the potential to develop as SCNT-treated embryos, microminipig-derived PEF clones termed as mMPEF were used as the nuclear donors. In a preliminary test, we found that nuclei from the transfected PEFs derived from a Clawn miniature pig as well as its intact parental cells were unable to promote development of SCNT embryos, probably due to their reduced developmental potential, which might have occurred during cell cultivation (data not shown). We, therefore, decided to use mMPEF as SCNT donors. We obtained a candidate clone (termed mMPEF-1) from microminipig-derived PEFs after transfection with seven PB vectors (five drug resistant and two fluorescent plasmids) and pTrans and subsequent selection with drugs. The microphotograph of mMPEF-1 is shown in Figure 3A. This clone had cells showing both red and green fluorescence and those with no fluorescence in a ratio of approximately 3:2. The latter cells may have all the drug resistance genes, but lack both fluorescent genes. SCNT using mMPEF-1 revealed that 18.1% (4/22) of the SCNT-treated embryos developed to normal blastocysts. As expected, 50% (2/4) of these embryos expressed both fluorescent markers (Figure 3B), but the other embryos did not fluoresce (data not shown). To confirm this event at the molecular biological level, they were singly isolated, lysed, and subjected to WGA. PCR analysis of these WGA samples demonstrated that blastocysts expressing both fluorescent markers had seven PB vectors (lanes one and two in Figure 3C), whereas those expressing no fluorescence lacked both fluorescent genes, but had five drug resistance genes (lanes three and four in Figure 3C). These results suggest that PB-mediated simultaneous integration of multiple constructs into the genome of porcine cells does not affect the development of cloned embryos.

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.