An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene

Jang, Goo; Bhuiyan, Mohammad Musharraf Uddin; Jeon, Hyun Yong; Ko, Kwang Hee; Park, Hee Jung; Kim, Min Kyu; Kim, Joung Ju; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk

doi:10.1016/j.theriogenology.2005.10.014

Cited by 33 publications

(29 citation statements)

References 49 publications

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“…Especially, somatic cell nuclear transfer (SCNT) provides an alternative, practical application and has been applied successfully to more than 20 animal species (Meissner and Jaenisch 2006). However, despite extensive efforts to improve the technique, the efficiency in terms of normal birth remains less than satisfactory (Jang et al 2006). In addition, a few reports have provided an effective and detailed upstream system to assess the promoter activity, construct the tissue-specific expression vector containing the dual selective markers, and prepare donor cell clones and transgenic embryos for SCNT.…”

Section: Introductionmentioning

confidence: 99%

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer

et al. 2014

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Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

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Section: Introductionmentioning

confidence: 99%

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer

et al. 2014

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“…Because Ca 2+ influx alone is not enough to activate embryos, sequential combination or single treatment with chemicals such as ionomycin, Ca-ionophore/6-DMAP and artificial electrical stimuli have been used as typical ways to activate nuclear transferred (NT) embryos [14,15], while cycloheximide and cytochalasin B have been used to activate NT embryos of certain species [16][17][18]. In the same way, many trials of new combinations of chemicals and adjustments of the activation time have been performed in various species [2,[19][20][21].…”

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confidence: 99%

Effects of Repetitive Ionomycin Treatment on <i>In Vitro</i> Development of Bovine Somatic Cell Nuclear Transfer Embryos

Kim¹,

Lee²,

Jeong³

et al. 2012

Journal of Reproduction and Development

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Abstract. To artificially activate embryos in somatic cell nuclear transfer (SCNT), chemical treatment with ionomycin has been used to induce transient levels of Ca 2+ and initiate reprogramming of embryos. Ca 2+ oscillation occurs naturally several times after fertilization (several times with 15-to 30-min intervals). This indicates how essential additional Ca 2+ influx is for successful reprogramming of embryos. Hence, in this report, the experimental design was aimed at improving the developmental efficiency of cloned embryos by repetitive Ca 2+ transients rather than the commonly used ionomycin treatment (4 min). To determine optimal Ca 2+ inflow conditions, we performed three different repetitive ionomycin (10 μM) treatments in reconstructed embryos: Group 1 (4-min ionomycin treatment, once), Group 2 (30-sec treatment, 4 times, 15-min intervals) and Group 3 (1-min treatment, 4 times, 15-min intervals). Pronuclear formation rates were checked to assess the effects of repetitive ionomycin treatment on reprogramming of cloned embryos. Cleavage rates were investigated on day 2, and the formation rates of blastocysts (BLs) were examined on day 7 to demonstrate the positive effect of repeated ionomycin treatment. In Group 3, a significant increase in BL formation was observed [47/200 (23.50%), 44/197 (22.33%) and 69/195 (35.38%) in Groups 1, 2 and 3, respectively]. Culturing embryos with different ionomycin treatments caused no significant difference among the groups in terms of the total cell number of BLs (164.3, 158.5 and 145.1, respectively). Additionally, expression of the anti-apoptotic Bcl-2 gene and MnSOD increased significantly in Group 3, whereas the expression of the pro-apoptotic Bax decreased statistically. In conclusion, the present study demonstrated that repeated ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis. Key words: Apoptosis, Bovine embryos, Ca 2+ oscillation, SCNT (J. Reprod. Dev. 58: [132][133][134][135][136][137][138][139] 2012) C a 2+ oscillation is a universal signaling cue that activates numerous cellular responses. Ca 2+ signaling is ubiquitous in some somatic cells and germ lines. In particular, in early embryo development, the fluctuating Ca 2+ concentration is a factor that brings about serial developmental mechanisms [1]. After fertilization, matured oocytes arrested in the metaphase stage of the second meiotic division (MII) turn into zygotes with subsequent signaling events such as depolarization, Ca 2+ oscillation, cortical reaction and pronucleus formation [2,3]. The exit from MII resumption and the further processes for forthcoming development do not occur before the concentration of intracellular Ca 2+ is increased by sperm penetration. Sperm is the initial signal for Ca 2+ release mediated by IP 3 (inositol trisphosphate), protein tyrosine kinases and PLC gamma (PLC-ζ in mammals), followed by reorganization of Ca 2+ -releasing machinery to trigger subsequent s...

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“…Somatic cell nuclear transfer and in vitro culture: The transfer of a donor fetal fibroblast into an enucleated oocyte was carried out as previously described [13]. Reconstructed embryos were electrically fused, activated for 4 min with ionomycin followed by culture for 4 hr in 6-DMAP.…”

Section: Methodsmentioning

confidence: 99%

Production of Transgenic Bovine Cloned Embryos Using Piggybac Transposition

Kim

Saadeldin

Choi

et al. 2011

The Journal of Veterinary Medical Science

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ABSTRACT. Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.KEY WORDS: bovine embryos, GFP, piggybac, RFP, SCNT.

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An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene

Cited by 33 publications

References 49 publications

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer

Effects of Repetitive Ionomycin Treatment on <i>In Vitro</i> Development of Bovine Somatic Cell Nuclear Transfer Embryos

Production of Transgenic Bovine Cloned Embryos Using Piggybac Transposition

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