Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (NeoR) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector

Uhm, Sang Jun; Kim, Nam‐Hyung; Kim, Teoan; Chung, Hyung Min; Chung, Kyunghwa; Lee, Hoon Taek; Chung, Kyuha

doi:10.1002/1098-2795(200012)57:4<331::aid-mrd4>3.0.co;2-7

Cited by 46 publications

(40 citation statements)

References 21 publications

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“…This result was in agreement with that in the previous studies, in which porcine fibroblasts [11,24] and bovine granulosa cells [1] were transfected with EGFP gene by a viral infection procedure and used as nuclear donor cells for NT.…”

Section: Discussionsupporting

confidence: 92%

“…TG sheep [14,20], cattle [4] and goats [9] were produced through the NT procedure by using the somatic cells transfected with specific foreign genes in combination with EGFP gene by using a lipofection procedure. Porcine TG embryos [11,18,24] and offspring [12,17] have been also produced through NT technology by using the somatic cells which were transfected with EGFP gene by a replicationdefective viral vector.The present study was conducted to verify the efficacy of the fusion/activation protocol of FAS with CHX treatment in the production of porcine TG embryos through NT procedure and to determine the effect of EGFP gene-transfection into the nuclear donor cells by a lipofection method on the …”

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confidence: 99%

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Effect of Fusion/Activation Protocol on In Vitro Development of Porcine Nuclear Transfer Embryos Constructed with Foreign Gene-Transfected Fetal Fibroblasts

Diaz

Mori

Nagano

et al. 2003

The Journal of Veterinary Medical Science

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ABSTRACT. The effect of fusion/activation protocol on in vitro development of porcine nuclear transfer (NT) embryos constructed with foreign gene-transfected somatic cells were investigated. NT embryos were produced by using enucleated M II oocytes and enhanced green fluorescence protein (EGFP) gene-transfected or non-transfected porcine fetal fibroblasts. One group of NT embryos received a single electrical pulse to induce fusion and activation simultaneously (FAS). The other group was fused 2 hr before activation (FBA) using two kinds of electrical pulses. Electrically activated NT embryos in both groups were treated with cycloheximide (CHX) before culture to assess the development to the blastocyst stage. After 6 days of culture, all morulae and blastocysts derived from EG FP-transfected fibroblasts emitted green fluorescence without mosaicism, and EGFP-gene product was also detected in all morulae and blastocysts examined. NT embryos undergoing FAS showed higher developmental capacity to blastocysts than those undergoing FBA, regardless of the EGFP transfection into the nuclear donor cells. The results also indicated that EGFP-gene transfection into nuclear donor cells has no obvious deleterious effect on the development of NT embryos to blastocysts. KEY WORDS: cycloheximide, green fluorescence protein, nuclear transfer, swine, transgenic embryo.J. Vet. Med. Sci. 65(9): 989-994, 2003 Transgenic (TG) farm animals have been routinely produced by pronuclear injection of foreign DNA into fertilized oocytes; however, embryo survival and transgene integration efficiencies are very poor [5]. Since the famous cloned sheep "Dolly" was born [27], nuclear transfer (NT) technology has become another methodology available for the production of TG animals. TG offspring derived from the NT embryos constructed with foreign gene-transfected somatic cells have been produced in sheep [20], cattle [4], goats [9] and pigs [17]. In the pig, however, somatic cell NT technology is still inefficient, and the factors affecting the success in the production of porcine NT embryos have to be explored.Somatic cell NT embryos have been most commonly produced by using enucleated oocytes at the metaphase II (M II) and various kinds of differentiated cells (nuclear donor cells) at the G0/G1 phase. A couplet of M II oocyte and nuclear donor cell is fused by adding an electric stimulation. Fused couplets are then subjected to an artificial activation to replace the fertilization process and to enter the first mitotic division. Fusion/activation process has been reported to critically affect the development of NT embryos. The NT embryos fused several hours before activation (termed 'FBA' in the present paper) showed higher development to the blastocyst stage than those fused and activated simultaneously (termed 'FAS' in the present paper) in mice [25], cattle [21,26] and pigs [11]. These observation indicated that prolonged exposure of transplanted nuclei at the G0/G1 stage to high M-phase promoting factor (MPF) activity may play an importan...

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 99%

Effect of Fusion/Activation Protocol on In Vitro Development of Porcine Nuclear Transfer Embryos Constructed with Foreign Gene-Transfected Fetal Fibroblasts

Diaz

Mori

Nagano

et al. 2003

The Journal of Veterinary Medical Science

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“…Similarly, GFP expression was not observed in all porcine transgenic NT embryos [17,18], and mosaic expression of GFP gene was shown in the embryos [18]. In contrast, GFP expression was observed in all transgenic NT blastocysts of bovine [1,3] and porcine [26] with no mosaic expression. The reason for mosaic expression of GFP gene observed in this study is not known at this moment.…”

Section: Discussionmentioning

confidence: 88%

Effect of Transfection and Passage Number of Ear Fibroblasts on In Vitro Development of Bovine Transgenic Nuclear Transfer Embryos

Bhuiyan

Cho

Jang

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. The objective of this study was to determine if the transfection of human prourokinase (ProU) gene and passage number of transfected ear fibroblasts affected in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human ProU was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker and human ProU gene into a pcDNA3 plasmid and transfected into bovine ear fibroblasts using a lipid mediated method. Abattoir derived oocytes were enucleated at 18-20 hr post maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the couplets were cultured in modified synthetic oviductal fluid (mSOF) medium for 168 hr. In Experiment 1, significantly lower rate in blastocysts formation (10.3%) was observed in transfected donor cells at early passage than that in nontransfected counterparts (22.1%, P<0.05). In Experiment 2, development to blastocysts and GFP expression in blastocysts were not significantly different between early (3-7) and late (8-12) passage donor cells (10.3 vs. 11.3% and 54.5 vs. 41.7%, respectively). This study indicates that in vitro development of bovine transgenic NT embryos is negatively influenced by transfection of human ProU gene into donor fibroblasts. However, passage number of transfected ear fibroblasts does not affect in vitro development of bovine transgenic NT embryos. KEY WORDS: bovine ear fibroblast, in vitro development, passage number, transfection, transgenic nuclear transfer embryo.

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“…This is the first report to describe the production transgenic rabbits by means of nuclear transfer. GFP or EGFP has previously been successfully used as a marker gene for transgenesis and gene expression (Takada et al 1997;Uhm et al 2000). Furthermore, donor cells expressing the GFP or EGFP have been used for nuclear transfer in mice (Eckardt et al 2005), pigs Hyun et al 2003), cattle (Bordignon et al 2003), and goats (Keefer et al 2001).…”

Section: Discussionmentioning

confidence: 99%

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

Li¹,

Guo

Shi

et al. 2008

Transgenic Res

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Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts were transfected with a pEGFP-C1 plasmid using lipofectamine() 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than 9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits.

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Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (NeoR) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector

Cited by 46 publications

References 21 publications

Effect of Fusion/Activation Protocol on In Vitro Development of Porcine Nuclear Transfer Embryos Constructed with Foreign Gene-Transfected Fetal Fibroblasts

Effect of Fusion/Activation Protocol on In Vitro Development of Porcine Nuclear Transfer Embryos Constructed with Foreign Gene-Transfected Fetal Fibroblasts

Effect of Transfection and Passage Number of Ear Fibroblasts on In Vitro Development of Bovine Transgenic Nuclear Transfer Embryos

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

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