Parthenogenetic embryo production in livestock provides headway to understand the role of the maternal and paternal genome during early embryonic development and offers an ethically competent source of embryonic stem cells. IGF2 and H19 are crucial imprinted genes and play an essential role in mammalian embryonic and placental development. The current study evaluated the methylation status of the DMR3 region of IGF2/H19 genes in goat blastocysts produced through parthenogenesis and in vitro fertilization (IVF) using bisulfite sequencing. For this, immature oocytes of good quality were subjected to in vitro maturation and subsequently used for parthenogenetic and IVF embryo production. The blastocyst rate was significantly (p < 0.05) higher for parthenogenetic embryos (21.11 +/- 2.51%) than for their IVF counterparts (8.23 +/- 1.02%). Furthermore, we analyzed the methylation status of the DMR3 region of the IGF2/H19 genes in both types of blastocysts. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (p 0.05) in parthenogenetic blastocysts as compared with IVF blastocysts (80.39 +/- 2.96, 32.55 +/- 4.37, respectively). We further analyzed the relative expression of IGF2/H19 in parthenogenetic and IVF blastocysts by qRT PCR. IGF2 (5.02-fold) gene was signifi-cantly upregulated, while H19 was significantly downregulated in parthenogenetic blastocyst compared to IVF blastocyst. The present study revealed a higher methylation status of the DMR3 region of IGF2/H19 in parthe-nogenetic blastocysts than the IVF blastocysts, which could upregulate IGF2 expression and the blastocyst rate.

Transgenic goats are ideal bioreactors for the production of therapeutic proteins in their mammary glands. However, random integration of the transgene within-host genome often culminates in unstable expression and unpredictable phenotypes. Targeting desired genes to a safe locus in the goat genome using advanced targeted genome-editing tools, such as transcription activator-like effector nucleases (TALENs) might assist in overcoming these hurdles. We identified Rosa 26 locus, a safe harbor for transgene integration, on chromosome 22 in the goat genome for the first time. We further demonstrate that TALEN-mediated targeting of GFP gene cassette at Rosa 26 locus exhibited stable and ubiquitous expression of GFP gene in goat fetal fibroblasts (GFFs) and after that, transgenic cloned embryos generated by handmade cloning (HMC). The transfection of GFFs by the TALEN pair resulted in 13.30% indel frequency at the target site. Upon cotransfection with TALEN and donor vectors, four correctly targeted cell colonies were obtained and all of them showed monoallelic gene insertions. The blastocyst rate for transgenic cloned embryos (3.92% ± 1.12%) was significantly (p < 0.05) lower than cloned embryos (7.84% ± 0.68%) used as control. Concomitantly, 2 out of 15 embryos of morulae and blastocyst stage (13.30%) exhibited site-specific integration. In conclusion, the present study demonstrates TALEN-mediated transgene integration at Rosa 26 locus in caprine fetal fibroblasts and the generation of transgenic cloned embryos using HMC.

Many contrasting reports are available on generation of bovine induced pluripotent stem cells (iPSCs) employing different timelines and culture conditions which signifies reprogramming process varies between species and cell types. The present study determines an optimum time period required to re-initiate reprogramming events in buffalo fibroblasts after introduction of exogenous genes (OCT4, SOX2, KLF4 and c-MYC) by lentiviral vector. The reprogramming efficiency is cumulative result of many factors including culture conditions and addition of growth factors in culture media. In our study, we observed when stem cell culture conditions were provided Day 5 post-transduction, it results in maximum reprogramming efficiency in comparison when same conditions were provided too early or on later days. The putative iPSCs were expanded on feeder layer for 15 passages and found positive for alkaline phosphatase and pluripotency markers (OCT4, SOX2, KLF4, c-MYC, UTF, TELOMERASE, FOXD3, REX1, STAT3, NUCLEOSTAMIN and TRA1-81). Also, they produced embryoid bodies showing expression for ectodermal (NF68, MOBP), mesodermal (ASA, BMP4) and endodermal (GATA4, AFP) markers to confirm their pluripotent nature. Our results suggest that reprogramming is accompanied by time dependent events and providing stem cell culture conditions at definite time during reprogramming can help in generation of iPSCs with greater efficiency.

Incorporating the gene of interest has always been keen interest to humans. Many potential application arise from transgenic animals, right from harvesting desired protein to creating species with enhanced ability. One such important enzyme Stearoyl Co-A Desaturase (SCD), which is involved in catalysis of monounsaturated fatty acids and polyunsaturated fatty acids from saturated fatty acids. It has also been implicated in diseases including obesity and the associated metabolic syndrome. Thus overexpressing buSCD in bovine species can really ameliorate human health, but the method of transfection has always been the limiting factor. In the present study, we have tried to study three non-viral methods: Nucleofection, Lipofection via Lipofectamine 3000 and Fugene HD. Initially, the somatic cell culture was established by culturing fetal ear skin (fetal fibroblast) explant in basal medium, comprising DMEM with 10% fetal bovine serum. To these somatic cells, our constructed vector, i.e. pAcGFPN1-buSCD was transfected using the above mentioned methods and non-transfected cell of same passage were used as control in the experiment. The efficiency was measured after 48–72 h of transfection via Fluorescence microscopy, Dye exclusion method (Trypan Blue-Live dead assay) and Real-time analysis. Each experiment was repeated 3 times and statistically analysed in Graphpad Prism. GFP expression in transfected Cells showed 62.4%± 2.3in nucleofection, 48.8% ±1.48 in Lipofectamine 3000 and50.7% ±2.48 in Fugene HD. Live-Dead assay revealed, cells transfected by nucleofection (76.42 ± 1.53) were significantly more alive (P >0.05) in comparison to Lipofectamine 3000 (67.34 ± 4.04)and Fugene HD(63.67 ± 1.53) respectively. Similar results were also shown by real-time analysis, in which apoptosis markers (CASPASE3 and p53) were significantly less expressive in nucleofected cells in comparison to cells transfected by Lipofectamine 3000 and Fugene HD. Overall, it can be inferred that nucleofection is better method of transfection as it shows lesser mortality and higher level of transfection in transfecting pAcGFPN1-buSCD, which can be further used for creating transgenic animals.

Recently augmentation of clinical and therapeutically important genes within the same species has become a subject of extensive research. Stearoyl-CoA desaturase (SCD) is a crucial enzyme converting saturated fatty acids to PUFAs and MUFAs and producing Conjugated Linoleic Acid (CLA) in its pathway, thus offering a complete package in granting nutritional values and immunity from fatal diseases. We aimed to identify the stable transfection method for transfecting a SCD construct in buffalo fetal fibroblast cells and determining its counter-effect on other cellular functions. Transfection of bovine SCD was performed using Nucleofection, Lipofectamine 3000 and Fugene HD. The level of transfection was analysed using flow-cytometry and efficiency was determined by MTT, relative proliferation, growth curve analysis and karyotyping. To understand the underlying genetic effect of transfection we studied the genes related to pluripotency (OCT4, SOX2, NANOG), epigenetics (DNMT1, DNMT3B, HDAC1), apoptosis (P53, CASPASE3, BAX, BID) and growth (G6PD, IGF1R). Considering the parameters tested a final transfection efficiency of 30% by nucleofection in buffalo fetal fibroblasts was observed, which was superior to all non-viral transfection methods tested to date. Also, it was observed that although all transfected cells showed higher level apoptosis, the level in nucleofected cells was the lowest. Overall when comparing the results of FACS, MTT assay and Gene expression profiling, it is suggested that nucleofection has a higher transfection efficiency and serves as a worthy candidate in delivering the expression vector with a comparatively lesser side effect on cellular functions and these cells may be further used for the production of transgenic buffalo.

Since the embryonic stem cells have knocked the doorsteps, they have proved themselves in the field of science, research, and medicines, but the hovered restrictions confine their application in human welfare. Alternate approaches used to reprogram the cells to the pluripotent state were not up to par, but the innovation of induced pluripotent stem cells (iPSCs) paved a new hope for the researchers. Soon after the discovery, iPSCs technology is undergoing renaissance day by day, i.e., from the use of genetic material to recombinant proteins and now only chemicals are employed to convert somatic cells to iPSCs. Thus, this technique is moving straightforward and productive at an astonishing pace. Here, we provide a brief introduction to iPSCs, the mechanism and methods for their generation, their prevailing and prospective applications and the future opportunities that can be expected from them.

Parthenogenesis has been observed in lower animals but no known instance has been reported in mammals because both maternal and paternal genomes are a fundamental prerequisite for embryogenesis. A major reason for developmental failure of uniparental zygotes is expression of certain genes in a parent-of-origin-specific manner, i.e. genomic imprinting of genes. Out of many imprinted genes identified so far, IGF2/H19 have been extensively studied and known to play an important role in fetal and placental development. Gene IGF2 is expressed by the paternal allele, H19 is transcribed from the maternal allele, and the reciprocal expression of both genes is regulated by the DMR3 region placed upstream of the H19 gene. In the present study we compared the methylation status of IGF2/H19 DMR in parthenogenetic activated (PA) and IVF goat (Capra hircus) blastocyst through bisulphite sequencing. For this, immature oocytes of usable quality were subjected to in vitro maturation and subsequently used for embryo production through parthenogenesis (n = 993) (by calcium ionophore and 6-DMAP activation) and IVF (n = 1096). It was found that embryo production rate at all the embryonic stages (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) was significantly higher (P < 0.05) in parthenogenesis (74.66 ± 3.35%, 61.90 ± 2.73%, 47.83 ± 2.95%, 38.13 ± 5.28%, and 21.11 ± 2.51%, respectively) as compared with IVF (55.21 ± 2.02%, 38.12 ± 2.48%, 28.53 ± 1.67%, 21.57 ± 1.59%, and 8.23 ± 1.02%, respectively). When blastocysts (n = 6 each) were subjected to TUNEL, it was found that PA blastocyst showed significantly higher (P < 0.05) total cell number (217.83 ± 18.80 v. 159.67 ± 13.94) and significantly low (P < 0.05) apoptotic index (2.04 ± 0.25 v. 4.03 ± 0.29) as compared with IVF blastocysts. For the methylation pattern study, we analysed 17 CpG sites on the DMR3 region of the IGF2/H19 gene. Variable methylation pattern was observed within these CpG sites in different clones (n = 15) of PA and IVF blastocyst. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (P < 0.05) in PA blastocysts as compared with IVF blastocysts (80.39 ± 2.96, 32.55 ± 4.37, respectively), which suggests higher expression of IGF2 in parthenotes. The result suggests IGF2 might play different roles in different species; the same expression pattern of IGF2 is observed in ovine, but a contrary result is found in porcine species. Our results signify the hypermethylation of IGF2/H19 DMR3, which leads to higher expression of IGF2 to support embryonic development at the blastocyst stage. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

Co-culture of pre-implantation embryos with oviducal epithelial cells mimics the in vivo conditions, thus, playing a crucial role in embryo metabolism and gene expression and finally supporting embryonic developmental competence in several ways. Hence, the objective of the present study was to evaluate the effect of goat oviducal epithelial cells (GOEC) co-culture on goat parthenogenetic embryonic development, quality, and relative mRNA abundance of genes related to developmental competence and oxidative stress. The GOEC were obtained from goat oviducts by squeezing and thorough washing with TCM-199 + 10% fetal bovine serum. Goat cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured in TCM-199 + 10% fetal bovine serum supplemented with 5 μg mL−1 of FSH, 10 μg mL−1 of LH, and 1 μg mL−1 of β-oestradiol for 27 h in CO2 incubator with 5% CO2 and at 38.5°C with >95% RH. In vitro matured cumulus–oocyte complexes were denuded and activated with 5 μM calcium ionophore and 2 mM 6-DMAP. Following activation, embryos were co-cultured with and without GOEC (control) in mCR2aa media. The blastocyst development rate was significantly (P < 0.05) higher (23.00 ± 1.15% v. 17.33 ± 1.45%) in the media cultured with GOEC than in control. The total cell number of blastocysts (n = 4) was also found to be significantly more (167.25 ± 17.51 v. 110.25 ± 12.02) than that of control (P < 0.05). However, the apoptotic index (3.76 ± 0.23% v. 7.97 ± 1.99%) was not significantly different in both groups. Further, RNA was isolated from both groups (20 each) of blastocysts on Day 8, and cDNA was prepared. Analysis by qPCR revealed that the relative mRNA abundance of development related genes, i.e. VEGF, BMP4, and CCNB1, showed significantly high (P < 0.05) expression, whereas the expression of CRABP1 was significantly low (P < 0.05) in GOEC co-culture than control. Oxidative stress related genes GPX-1 and SOD2 had comparable expression in both the culture systems, whereas a nonsignificant (P < 0.05) increase in expression of PRDX1 was observed in GOEC co-culture group. In conclusion, co-culture of embryos with GOEC in the simple culture media like mCR2aa helps in improving developmental competence and quality of parthenogenetic embryos. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.