22 PRODUCTION OF PORCINE TRANSGENIC CELL LINE INSERTED WITH SV40LT, EGFRvIII Gene, AND INDUCIBLE CreERT SYSTEM

2016 ◽  
Vol 28 (2) ◽  
pp. 141
Author(s):  
S. U. Hwang ◽  
J. D. Yoon ◽  
K. Y. Eun ◽  
H. G. Kim ◽  
S. H. Hyun
Keyword(s):  
Cell Line ◽  
Embryo Development ◽  
Donor Cell ◽  
Disease Models ◽  
Blastocyst Formation ◽  
Cooperative Research ◽  
Normal Cells ◽  
Transgenic Cell ◽  
Significant Difference ◽  
Transgenic Cell Line

Transgenic pigs are currently believed to be an important model for biomedical research, including for disease models, pharmaceutical toxicity testing, and regenerative medicine. However, production efficiency of animal disease models using somatic cell NT (SCNT) is very low. One of the main reasons is probably characteristics of the transgene. In this study, we introduce SV40LT oncogene into the fibroblast cells in order to establish immortalized transgenic cell line for producing the pig model of human brain cancer. We evaluated the effect of SV40LT oncogene on transgenic SCNT embryo development. As a results, the cleavage rates (73.8 ± 4.0 and 48.6 ± 2.4 in the normal and SV40LT group, respectively; P < 0.05) and blastocyst formation rates (19.5 ± 1.2 and 5.6 ± 1.8 in the normal and SV40LT group, respectively; P < 0.05) of transgenic SCNT embryos was significantly lower than the case of using normal cells. In addition, we evaluated the transgenic SCNT embryo development of the donor cell transfected with SV40LT and HrasV12 genes (SV40LT+HrasV12 group). As a results, there was no significant difference between the groups in the cleavage rates, but blastocyst formation rates of transfected SCNT embryos (SV40LT+HrasV12 group) was significantly lower than the case of using normal cells (19.5 ± 1.2 and 6.2 ± 1.8 in the normal and SV40LT+HrasV12 group, respectively; P < 0.05). Genes SV40LT or HrasV12 showed a negative effect on SCNT cloned embryo development. Therefore, a Cre/loxP inducible system was applied to producing donor cells transfected with EGFRvIII and SV40LT gene. As a result, the cleavage rates (73.8 ± 4.0 and 68.6 ± 6.6 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) and blastocyst formation rate (19.5 ± 1.2 and 23.0 ± 3.7 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) were improved to the same level, when used as a donor cell to a normal cell. In conclusion, these results indicated that harmful effects of transgenic SCNT embryo development caused by the characteristics of the inserted genes can be overcome through the inducible system. Further studies are needed to experiment with mRNA expression of apoptotic gene and target gene in 4- to 8-cell embryos and blastocysts. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean government (NRF-2013R1A2A2A04008751), Republic of Korea.

scholarly journals 22PRODUCTION OF A CLONED CALF USING KIDNEY CELLS OBTAINED FROM A 48-HOUR COOLED CARCASS

2004 ◽  
Vol 16 (2) ◽  
pp. 133 ◽  
Author(s):  
A.M. Adams ◽  
S.L. Pratt ◽  
J.R. Gibbons ◽  
S. Arat ◽  
D.S. Respess ◽  
...  
Keyword(s):  
Cell Line ◽  
Embryo Development ◽  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Donor Cell ◽  
Kidney Cells ◽  
Tissue Samples ◽  
Significant Difference ◽  
Beef Heifer ◽  
Beef Carcass

The ability to produce cloned livestock using postmortem tissue could incorporate an additional application into the field of nuclear transfer. This study examined the feasibility of producing cloned cattle using a primary cell line established from a postmortem beef carcass. A market beef heifer processed at a USDA-certified slaughterhouse was used to develop a primary somatic cell line. Tissue samples were taken from the kidney and forelimb regions either 1) immediately following slaughter (fresh) or 2) 48h postslaughter (cooled) where the carcass was housed at 2 to 4°C. Tissue was removed and placed on ice in PBS+5.0% (v:v) penicillin/streptomycin. A primary culture was established using standard techniques and cultured in supplemented DMEM F-12 medium. Once established, cells were trypsinized and either frozen or continually passaged. Cells used for nuclear transfer (NT) were passaged (48h before use) and cultured with 15μM roscovitine roughly 24h prior to nuclear transfer. Cells were approximately 80% confluent and between passage numbers 1 and 11 at the time of NT. Selected slaughterhouse-derived oocytes were matured in supplemented TCM 199 medium for 18–20h at 39°C in 5.0% CO2 and air. Mature Metaphase II oocytes were vortexed and stained with Hoechst 33342 to help with chromatin removal. Following enucleation, roscovitine-treated carcass cells were placed in the perivitelline space of the oocyte. Reconstructed NT embryos were fused in Zimmermann’s medium and pulsed using needle-like electrodes. This was followed by activation using a combination of calcium ionophore (5μM), cytochalasin D (5μgmL−1), and cycloheximide (10μgmL−1) in TCM+10% FBS. Fused NT embryos were cultured in 50-μL drops of BARC medium (USDA, Beltsville, MD) for 7 days at 39°C in a 5% CO2, 5% O2 and 90% N2 environment. Embryo development for all four groups (Table 1) was assessed with blastocysts (grade 1 or 2) being transferred into recipient cows 7 days post-estrus. Cleavage rates were not significantly different between groups, and the use of either fresh or cooled cells did not impact blastocyst formation. However, there was a significant difference (P=0.05) in % blastocyst based on the source of the donor cell. Overall, one live calf resulted from 34 transferred NTs produced using kidney cells taken from a 48h cooled carcass. These results display the feasibility of producing cloned calves from cells collected post mortem, which ultimately could be used as a tool to select breeding bulls based on their own steer carcass characteristics. Table 1 Embryo development and pregnancy data for the production of beef carcass clones

scholarly journals Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

1998 ◽  
Vol 116 (2) ◽  
pp. 853-857 ◽  
Author(s):  
Serap Whitmer ◽  
Camilo Canel ◽  
Didier Hallard ◽  
Cecilia Gonçalves ◽  
Robert Verpoorte
Keyword(s):  
Cell Line ◽  
Catharanthus Roseus ◽  
Transgenic Cell ◽  
Transgenic Cell Line

scholarly journals Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection

PLoS ONE ◽  
2016 ◽  
Vol 11 (5) ◽  
pp. e0154238 ◽  
Author(s):  
Linlin Zhang ◽  
Zhengzhi Cui ◽  
Lei Zhou ◽  
Youmin Kang ◽  
Li Li ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cell Line ◽  
High Efficiency ◽  
Respiratory Syndrome Virus ◽  
Transgenic Cell ◽  
Transgenic Cell Line

20 Aggregation of yak heterospecific somatic cell nuclear transfer embryos improves cloning efficiency

2020 ◽  
Vol 32 (2) ◽  
pp. 135
Author(s):  
M. Yauri Felipe ◽  
M. Duque Rodríguez ◽  
A. De Stéfano ◽  
D. Salamone
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Formation Rate ◽  
Donor Cell ◽  
Blastocyst Formation ◽  
Fluid Medium ◽  
Exact Test ◽  
Significant Difference

Cloning endangered species has the limitation that generally the number of available oocytes is limited. Reprogramming the nuclei heterospecifically using an enucleated oocyte from a different species is an alternative. Aggregation of SCNT (somatic cell nuclear transfer) embryos from the same specie results in improved embryo development. However, after aggregation of heterospecific SCNT embryos from different genera, no effects were observed (Moro et al. 2015 Reproduction 50, 1-10). The objective of this study was to evaluate the influence of aggregation of yak (Bos grunniens) embryos produced by heterospecific SCNT using enucleated oocytes from an animal from the same genus Bos taurus. As control homospecific SCNT of Bos taurus, parthenogenic zone-free embryos and IVF embryos were used. Cumulus-oocyte complexes were recovered from bovine slaughterhouse ovaries by follicular aspiration. The cumulus-oocyte complexes were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10μgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 22h, at 6.5% CO2 in humidified air and 38.5°C. After denudation, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation. Staining was performed with Hoechst 33342 to observe MII. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with the donor cell followed by electrofusion. All reconstituted embryos were activated using ionomcine. This was followed by a treatment with 6-dimethylaminopurine for 3h. Zona-free reconstituted cloned embryos were cultured in the wells of the well system, placing one (1×) or two (2×) per microwell, in synthetic oviductal fluid medium. The experimental groups were parthenogenic zone free; IVF; reconstituted embryos bull fibroblast-enucleated oocyte from cow (BC1×); reconstituted embryos yak fibroblast-enucleated oocyte from cow (YC1×); and reconstituted embryos aggregated yak fibroblast-enucleated oocyte from cow (YC2×). In all experimental groups, cleavage of at least one embryo in the wells and blastocyst formation at Day 7 were assessed. The effect of cloned embryo aggregation on blastocyst rates was analysed using Fisher exact tests (GraphPad Prisma 8), and results are shown on Table 1. Results demonstrated that aggregation of two SCNT heterospecific embryos increased the blastocyst formation rate of yak (P&lt;0.05). In conclusion aggregation in yak heterospecific SCNT embryos from species of the same genus (Bos) can improve development to blastocyst. Table 1.Aggregation of yak heterospecific somatic cell nuclear transfer embryos Experimental group1 No. of embryos No. of embryos-wells2 Cleavage (%) Blastocyst (%) PZF 68 68 66 (97.06%)a 17 (25.00%)acd IVF 89 - 81 (91.01%)ab 39 (43.82%)b BC1× 45 45 41 (91.11%)b 6 (13.33%)cd YC1× 101 101 77 (76.24%)c 14 (13.86%)c YC2× 134 67 61 (91.04%)ab 21 (31.34%)ab a-dDifferent superscripts in the same column indicate significant difference (Fisher's exact test, P&lt;0.05). 1PZF, parthenogenetic zone free; IFV, IVF fecundation; BC1×, clone of bovine; YC1×, clone of yak-bovine; YC2×, clone of yak-bovine added. 2Wells used with embryos.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 222
Author(s):  
S. H. Lee ◽  
E. M. N. Setyawan ◽  
B. C. Lee
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Significant Difference ◽  
Epidermal Growth ◽  
Oviduct Cell

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P < 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

scholarly journals Correction: Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection

PLoS ONE ◽  
2016 ◽  
Vol 11 (7) ◽  
pp. e0160325 ◽  
Author(s):  
Linlin Zhang ◽  
Zhengzhi Cui ◽  
Lei Zhou ◽  
Youmin Kang ◽  
Li Li ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cell Line ◽  
High Efficiency ◽  
Respiratory Syndrome Virus ◽  
Transgenic Cell ◽  
Transgenic Cell Line
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