Ramakant Kaushik

Isolation, characterization, and EGFP expression in the buffalo (Bubalus bubalis) mammary gland epithelial cell line

This study was aimed to establish a buffalo mammary epithelial cells (BuMECs) line and maintain it for long-term by subculturing. BuMECs isolated from lactating buffalo mammary glands were cultured on a collagen matrix gel. BuMECs expressed significant amounts of the epithelial cell specific marker cytokeratin 18 as determined by immunohistochemistry. The BuMECs displayed monolayer, cobble-stone morphology, and formed lumen-, dome-, and duct-like structures. Furthermore, they were capable of synthesizing CSN2, BLG, ACACA, and BTN1A1, showed viability after thawing and expressed milk protein genes. The enhanced green fluorescent protein gene was transferred successfully into the BuMECs using lipofection method and the transfected cells could be maintained for long-term in culture by subculturing.

WNT3A signalling pathway in buffalo (Bubalus bubalis) embryonic stem cells.

The aim of this study was to investigate the transcriptional profile and role of WNT3A signalling in maintaining buffalo embryonic stem (ES) cells in a pluripotent state and in the induction of their differentiation. ES cells were derived from embryos produced by in vitro fertilisation (iESC), parthenogenesis (pESC) and hand-made cloning (cESC). The expression of WNT3A, its receptors and intermediate signalling pathways were found to be conserved in ES cells derived from the three different sources. WNT3A was expressed in ES cells but not in embryoid bodies derived from iESC or in buffalo fetal fibroblast cells. It was revealed by real-time polymerase chain reaction analysis that following supplementation of culture medium with WNT3A (100, 200 or 400ngmL(-1)) a significant increase (P<0.05) was observed in the expression level of β-CATENIN, which indicated the activation of the canonical WNT pathway. WNT3A, in combination with exogenous fibroblast growth factor-2 and leukaemia inhibitory factor, induced proliferation of undifferentiated ES cells. Differentiation studies showed that WNT3A caused formation of scaffold-like structures and inhibition of differentiation into neuron-like cells. In conclusion, the WNT3A signalling pathway is necessary both for maintaining undifferentiated buffalo ES cells as well as for directing their differentiation.

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization

In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus-oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus-oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus-oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.

Quantitative expression of pluripotency-related genes in parthenogenetically produced buffalo (Bubalus bubalis) embryos and in putative embryonic stem cells derived from them.

Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells.

209 PRODUCTION OF BUFFALO (BUBALUS BUBALIS) EMBRYOS CONTAINING HUMAN LYSOZYME GENE

Lysozyme is a ubiquitous enzyme found in all major taxa of living organisms with a diverse role in human and animal health. It plays an important role as a nonspecific immune factor and anti-inflammatory factor and is a part of the innate immune system. Research on the expression of recombinant human lysozyme is, thus, potentially valuable to the dairy industry. Therefore, the present study was carried out to observe the developmental competence and quality of cloned embryos containing the human lysozyme gene. Primary fetal fibroblast cells were obtained from a slaughterhouse-derived fetus. The initial passage cells were transfected with pAchLYZ vector containing human lysozyme gene and green fluorescence protein (GFP) via nucleofection, lipofectamine, and FuGene (Roche Diagnostics, Indianapolis, IN, USA). Transfected cells were selected by adding G418 (400-800µgµL-1), which selectively killed the nontransfected cells in culture. The presence of hLYZ gene in transfected cells was confirmed by PCR amplification of this gene. For cloned embryo production, reconstructs were formed with 2 enucleated demi oocyte fused with 1 donor cell of hLYZ transfected cells or nontransfected fibroblast cells. Gene expression in the resulting embryos was assessed for apoptosis (BID, BAX, and BCL-XL) and development- (G6PD, IGF1R, and FGF) and pluripotency-related (OCT4, SOX2, and NANOG) genes. The transfection efficiency of the cells by nucleofection methods was the highest and toxicity to the cells was minimum as compared with chemical methods. Transfected cells expressed GFP within 48 to 72h of transfection. When these cells were passaged the intensity of GFP expression was reduced and the reduced level was maintained in subsequent passages. A total of 114 and 60 reconstructed embryos were produced using transfected and nontransfected cells, respectively. When transfected cells were used, the cleavage, 4 cell, 8 to 16 cell, morula, and blastocyst rate was 62.01±4.78, 43.54±3.93, 27.69±4.44, 24.55±5.12, and 22.87±5.39%, respectively, whereas rates were 73.34±2.36, 68.34±4.82, 59.89±2.39, 46.44±2.64, and 36.36±2.53% for nontransfected cells, respectively. The cleavage rate had no significant (P<0.05, Students t-test) difference in both type of donor cells but all other stages [i.e. 4 cell, 8 to 16 cell, morula, and blastocyst rate was significantly (P<0.05) lower with transfected cells]. Most of the transgenic cloned embryos expressed GFP and integrated with hLYZ gene. We found that proapoptotic gene (BID and BAX) expression did not differ, whereas BCL-XL expression was significantly low (P<0.05) in transgenic embryos. The development- (G6PD, IGF1R, and FGF) and pluripotency-related (OCT4, SOX2, and NANOG) gene expression was significantly (P<0.05) lower in transgenic embryos. In conclusion, transgenic cloned embryo successfully developed up to the blastocyst stage in the preliminary study for producing genetically modified animal with human milk components having antimicrobial activity, which would be potentially valuable for human as well as animal health.

208 PRODUCTION OF TRANSGENIC CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS CONTAINING HUMAN INSULIN GENE THROUGH HAND-GUIDED CLONING

Diabetes is a growing disease worldwide and has emerged as a major healthcare problem in India. Insulin is an essential medicine for the treatment of diabetes. Large dairy animals, such as buffaloes and cows, may be used as bioreactors for cost-effective production of human insulin. The present study was aimed to produce transgenic buffalo embryos containing the human insulin gene through hand-guided cloning for production of transgenic animals. Buffalo female fetal fibroblast cells at passage number 3 were transfected using mammary gland- specific expression vector containing the human insulin gene under buffalo β-lactoglobulin promoter by nucleofection method and cultured with G418 drug for 3 weeks to obtain positive transgenic cell clones. Transgene integration into buffalo female fetal fibroblast genome was confirmed by PCR and Southern blotting. Nontransfected and transgene integrated cells were used as nuclear donors to produce embryos by the hand-guided cloning technique. The developmental competence and quality of embryos as judged by total cell number and TUNEL assay were compared among transgenic and nontransgenic (control) embryos. The blastocyst rate was lower (P<0.05) for transgenic embryos than that of nontransgenic cloned embryos (35.97±2.16v. 45.80±4.11, respectively). The apoptotic index was found to be lower (P<0.05) for control blastocysts than that for transgenic blastocysts. However, the total cell number was similar (P<0.05) among transgenic and control cloned blastocysts. Thus, transgenic cells, and subsequently transgenic embryos containing the human insulin gene, were successfully produced and transferred in recipients. In the future, these may be used for production of transgenic buffalo expressing human insulin in its milk and thus can be further utilised in large-scale production of human insulin.