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Pandey A.,Transgenic Research Laboratory | Gupta S.C.,Transgenic Research Laboratory | Gupta N.,Transgenic Research Laboratory
Cellular Reprogramming

To improve the efficiency of somatic cell nuclear transfer (SCNT)-derived embryos in buffaloes (Bubalus bubalis), skin fibroblast, cumulus, and granulosa cells were cultured up to the 15th passage and cloned embryos were produced from each cell type. At the 15th passage the cumulative population doublings (CPDs) in cumulus cells was higher (60.78) than skin fibroblast (57.12) and granulosa (56.05) cell lines. Gene expression of chromatin remodelling proteins, that is, HDAC1, DNMT1, DNMT3a, and DNMT3b, were comparable at all five passages (P-3, P-6, P-9, P-12, and P-15) groups in cumulus cells but different in skin fibroblast and granulosa cells. Cleavage and blastocyst production rate in cumulus (65.9 and 27.4%)-derived embryos was higher than skin fibroblast (63.8 and 24.3%) and granulosa (62.5 and 22.3%)-derived embryos. Expressions of HDAC1, DNMT1, and DNMT3a mRNA in cumulus-derived blastocysts were similar to IVF blastocysts (control), whereas skin fibroblast and granulosa-derived blastocysts expression was significantly different (p≤0.05). DNMT3b mRNA expression in all the three donor cell types and IVF control were similar. The expression pattern of these genes showed the effect of donor cell type with different epigenetic reprogramming capabilities for SCNT embryo production rate. Overall, results indicated that cumulus cells are the best nuclear donor for SCNT. Copyright 2010, Mary Ann Liebert, Inc. Source

Pandey A.,Transgenic Research Laboratory | Gupta S.C.,Transgenic Research Laboratory | Gupta N.,Transgenic Research Laboratory

Summary Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are commonly added to maturation media to improve cumulus expansion known to be a predictor of oocyte maturation. Therefore, effects of various concentrations of FSH (1000 ng/ml), LH (1000 ng/ml) and FSH + LH (1000 ng/ml each) in comparison with control (without FSH + LH) cultured oocytes were investigated. FSH and LH (1000 ng/ml each) induced significantly more cumulus expansion and polar body numbers, as compared with control and treatments of 1000 ng/ml FSH and 1000 ng/ml LH alone. Expression of FSH receptor (r), LHr and Cx43 mRNAs was determined by real-time PCR in cumulus-oocyte complexes (COCs) and denuded oocytes at different maturation times. Expression of all three genes was higher in COCs compared with denuded oocytes, confirming the importance of cumulus cells in oocyte maturation. FSHr and connexin 43 (Cx43) mRNA abundance in both COCs and denuded oocytes was highest at 0-6 h of maturation and decreased subsequently. However, LHr mRNA abundance increased from 6 h up to 24 h of maturation. The study concluded that FSH, LH receptors and Cx43 gene expression regulation is an index related to oocyte maturation. Copyright © 2010 Cambridge University Press. Source

Pandey A.,Transgenic Research Laboratory | Pandey A.,Guru Jambheshwar University of Science and Technology | Gupta S.C.,Transgenic Research Laboratory | Singh N.,Guru Jambheshwar University of Science and Technology | And 2 more authors.
Reproduction in Domestic Animals

Growth factors in culture media are known to affect the embryo production rates in in vitro production cultures. To improve the efficiency of somatic cell nuclear transfer (SCNT) derived embryos in Indian buffaloes (Bubalus bubalis), embryos were cultured in three different culture mediums viz. Group-A; TCM-199 + FBS, Group-B; TCM-199 + Poly vinyl alcohol (PVA) and Group-C; CR1aa + BSA. Embryo production rate and expression level of insulin-like growth factor genes (IGF-1, IGF-1R, IGF-2 and IGF-2R) were analysed in embryo culture. Cleavage and blastocyst production rates were 62.5% and 22.3% in Group-A, 53.8% and 13.0% in Group-B and 62.0% and 19.2% in Group-C respectively, whereas in in vitro fertilization (IVF) control cultured in TCM-199 plus 10% FBS, rates were 79.1% and 29.4%. Relative gene expression of SCNT embryos was compared with that in IVF control. IGF-1 and IGF-2 mRNA expression at blastocyst stage was up-regulated (p ≤ 0.05) in all culture groups, while IGF-1R and IGF-2R expression was down regulated (p ≤ 0.05) in Group-B and Group-C. In conclusion, the higher mRNA levels at certain stages in different culture conditions affected in vitro development of SCNT embryos. These results show that the transcript level of the insulin-like growth factor genes was significantly altered by in vitro culture condition. Culture medium TCM-199 with 10% FBS produced higher number of embryos and was able to co-op with gene expression of IVF control. Differences continue to be observed between SCNT cultured and IVF embryos, and until these differences are minimized, aberrations in SCNT embryonic development will continue to arise. © 2009 Blackwell Verlag GmbH. Source

Choudhary S.,Transgenic Research Laboratory | Ahlawat S.P.S.,Transgenic Research Laboratory | Gupta S.C.,Transgenic Research Laboratory | Gupta N.,Transgenic Research Laboratory
Indian Journal of Animal Research

Experiment was carried out at National Bureau of Animal Genetic Resources, Kamal to identify polymorphisms of the prion protein gene (PrP) at the codons (136,154 and 171) responsible for the susceptibility and resistance of the scrapie disease in the sheep. Blood samples of 50 animals of the Garole sheep breed were collected from its native tract in and around South 24 Pargana district of West Bengal from the eastern region. PCR amplicons of approximate size 358 bp were generated for PrP gene exon 3 partial CD's covering 352 to 700 nucleotide positions. Genotype ARQ/ARQ was found in the analyzed population with the 100% frequency. SNP at codon 127 and 189 are GG/GS and QQ/QL with frequencies of 72.73/27.27 and 90.91/9.09 respectively. In all flocks, ARQ/ARQ genotype was the dominant haplotype regardless of sheep age, sex and health. However, the generated data will help for the detection of SNP markers related to disease resistance or susceptibility in India. Source

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