Production of Water Buffalo SCNT Embryos by Handmade Cloning.

Cloning by somatic cell nuclear transfer (SCNT) involves the transfer of a somatic nucleus into an enucleated oocyte followed by chemical activation and embryo culture. Further, handmade cloning (HMC) is a simple and efficient SCNT method for large-scale embryo production. HMC does not require micromanipulators for oocyte enucleation and reconstruction since these steps are carried out using a sharp blade controlled by hand under a stereomicroscope. In this chapter, we review the status of HMC in the water buffalo (Bubalus bubalis) and further describe a protocol for the production of buffalo-cloned embryos by HMC and assays to estimate their quality.

Comparison the effects of 5-Aza-2'-deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos.

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.

MeDIP-sequencing for profiling global DNA methylation in buffalo embryos produced by in vitro fertilization.

Assisted reproductive technique like in vitro fertilization has contributed immensely in producing genetically improved livestock. Production of embryos under in vitro conditions can affect global DNA methylation pattern during the course of embryonic development. The present study is aimed at the generation and comparison of global DNA methylome of embryos at 2-cell, 8-cell and blastocyst stage of buffalo embryos produced by in vitro fertilization using MeDIP-Sequencing. It is observed that there is a profound difference in the global DNA methylation profile of IVF embryos at different developmental stages. These differences are manifested throughout the course of embryonic development. Pathways like Wnt signaling pathway, gonadotropin-releasing hormone receptor pathway and integrin signaling were found to be majorly affected by hypermethylation of DNA in IVF embryos throughout the development.

Global MicroRNA Expression Profiling of Buffalo (Bubalus bubalis) Embryos at Different Developmental Stages Produced by Somatic Cell Nuclear Transfer and In-Vitro Fertilization Using RNA Sequencing.

Despite the success of cloning technology in the production of offspring across several species, its application on a wide scale is severely limited by the very low offspring rate obtained with cloned embryos. The expression profile of microRNAs (miRNAs) in cloned embryos throughout embryonic development is reported to deviate from regular patterns. The present study is aimed at determining the dynamics of the global expression of miRNA profile in cloned and in-vitro fertilization (IVF) pre-implantation embryos at different developmental stages, i.e., the two-cell, eight-cell, and blastocyst stages, using next-generation sequencing. The results of this study suggest that there is a profound difference in global miRNA profile between cloned and IVF embryos. These differences are manifested throughout the course of embryonic development. The cloned embryos differ from their IVF counterparts in enriched Gene Ontology (GO) terms of biological process, molecular function, cellular component, and protein class categories in terms of the targets of differentially expressed miRNAs. The major pathways related to embryonic development, such as the Wnt signaling pathway, the apoptosis signaling pathway, the FGF signaling pathway, the p53 pathway, etc., were found to be affected in cloned relative to IVF embryos. Overall, these data reveal the distinct miRNA profile of cloned relative to IVF embryos, suggesting that the molecules or pathways affected may play an important role in cloned embryo development.

Global DNA methylation profiles of buffalo (Bubalus bubalis) preimplantation embryos produced by handmade cloning and in vitro fertilization.

Somatic cell nuclear transfer technique (SCNT) has proved to be an outstanding method of multiplication of elite animals but accompanied with low efficiency and live birth rate of cloned animals. Epigenetic alterations of DNA has been one of the culprits behind this issue. Cloned embryos are found to deviate slightly from regular pattern of demethylation and re-methylation at the time of nuclear reprogramming and embryonic development when compared with embryos produced by in vitro fertilization (IVF). Thus, the present study was aimed at evaluating global DNA methylation profiles of cloned embryos at 2-cell, 8-cell and blastocyst stages and compare it with corresponding stages of embryos produced by IVF by using MeDIP-Sequencing on Illumina-based platform. We found out that cloned embryos exhibited significantly different DNA methylation pattern as compared to IVF embryos with respect to distribution of differentially methylated regions in different components of genome, CpG islands distribution and methylation status, gene ontological profiles and pathways affected throughout the developmental stages. The data generated from MeDIP-Seq was validated at blastocyst stage cloned and IVF embryos by bisulfite-sequencing PCR on five randomly selected gene regions.

Exposure to Pulsed Electromagnetic Fields Improves the Developmental Competence and Quality of Somatic Cell Nuclear Transfer Buffalo (Bubalus bubalis) Embryos Produced Using Fibroblast Cells and Alters Their Epigenetic Status and Gene Expression.

We examined the effects of treatment with pulsed electromagnetic fields (PEMFs) on cumulus cells and buffalo somatic cell nuclear transfer (SCNT) embryos. PEMF treatment (30 µT for 3 hours) of cumulus cells increased (p < 0.05) the relative cell viability and cell proliferation and the expression level of OCT4, NANOG, SOX2, P53, CCNB1, and GPX, but decreased (p < 0.05) that of DNMT1, DNMT3a, GSK3b, and BAX, whereas the expression level of DNMT3b, GLUT1, BCL2, CASPASE3, SOD1, and CATALASE was not affected. PEMF treatment of SCNT embryos at the beginning of in vitro culture increased (p < 0.05) the blastocyst rate (51.4% ± 1.36% vs. 42.8% ± 1.29%) and decreased (p < 0.01) the apoptotic index to the level in in vitro fertilization blastocysts, but did not significantly alter the total cell number and the inner cell mass:trophectoderm cell number ratio of blastocysts compared to the controls. PEMF treatment increased the expression level of NANOG, SOX2, CDX2, GLUT1, P53, and BCL2 and decreased that of BAX, CASPASE3, GSK3b, and HSP70, but not OCT4, DNMT1, DNMT3a, DNMT3b, HDAC1, and CCNB1 in blastocysts. It increased (p < 0.001) the global level of H3K27me3 but not H3K18ac. These results suggest that PEMF treatment of SCNT embryos improves their developmental competence, reduces the level of apoptosis, and alters the expression level of several important genes related to pluripotency, apoptosis, metabolism, and stress.

Production of Transgenic Handmade Cloned Goat (Capra hircus) Embryos by Targeted Integration into Rosa 26 Locus Using Transcription Activator-like Effector Nucleases.

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.

Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts.

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.

Optimization and Comparison of Three-Dimensional Culture Conditions in Different Media of Coculture and Encapsulation System for In Vitro Follicular Development in Bubalus bubalis.

The establishment of an in vitro culture system for complete oocyte maturation from the early stages of ovarian follicles is still a challenge. The aim of the present study was to assess the effect of different matrix with different culture media on the developmental growth of ovarian follicles in vitro. An ovarian histoarchitectural study was carried out to identify the primordial (0.027-0.039 mm), primary (0.041-0.079 mm), small preantral (0.085-0.131 mm), large preantral (0.132-0.294 mm), small antral (0.387-0.589 mm), and large antral (1.188-1.366 mm) follicles. Thus, large preantral follicles (0.2-0.3 mm) were mechanically isolated and cultured subsequently in different microconditions such as Dulbecco's modified Eagle's medium, Tissue Culture Medium-199 (TCM-199) and Opti-minimum essential medium, with same supplements where control (without matrix) was compared with matrix (coculture and encapsulation), which includes (1) buffalo fetal fibroblast cells, (2) cumulus cells, (3) ovarian mesenchymal cells, (4) collagen, (5) gelatin, and (6) Matrigel, cultured for 7 days in CO2 incubator at 38.5°C (5% CO2 in air). Cultured follicles were evaluated for growth rate (107.88% ± 10.24%), maturation rate (51.06% ± 6.53%), survivability rate (56.52% ± 3.42%), and antioxidant (catalase; CAT [1.58 ± 0.04 U/mg], superoxide dismutase; SOD [4.63 ± 0.05 U/mg], lactate dehydrogenase; LDH [1.48 ± 0.01 U/mg]) enzymatic activities, which showed significantly (p < 0.05) positive results in growth model with media TCM-199 than other studied groups. Furthermore, the development of large preantral follicles augmented significantly (p < 0.05) for growth rate (248.54% ± 9.51%), maturation rate (75.81% ± 7.07%), survivability rate (81.82% ± 3.02%), antioxidant (CAT [2.05 ± 0.03 U/mg], SOD [3.13 ± 0.12 U/mg], LDH [2.55 ± 0.51 U/mg]), and estradiol (175.83 ± 5.92 pg/mL) activities when they were encapsulated in Matrigel with nutritional requirements fulfilled by media TCM-199. These results provide better insight for the optimization of culture conditions for in vitro follicular development in the water buffalo, which will eventually assist in resolving the limitation of obtaining fewer competent oocytes for the embryo production in the species.

Effect of basic fibroblast growth factor (FGF2) on cumulus cell expansion, in vitro embryo production and gene expression in buffalo (Bubalus bubalis).

The present study was undertaken to evaluate the effect of different concentration of FGF2 viz. 5 ng (T1), 10 ng (T2), and 20 ng/mL (T3) on cumulus cell expansion, oocyte maturation, in vitro embryo production, total cell number (TCN) of the blastocyst, and expression of the FGF2 and FGFR2 transcripts in buffalo oocytes and the embryos. Results showed that the effect of FGF2 on the diameter of buffalo COC was significantly higher (P < 0.05) in the T1 group than the other groups at 24h of maturation. The maturation and cleavage rate of oocytes was significantly higher (P < 0.05) in the T3 group than the control, however, the values did not different (P> 0.05) from other groups. The effect of FGF2 on morula and blastocyst yield did not different (P > 0.05) between treatment groups. However, the TCN of the blastocyst was slightly higher (P > 0.05) in the T3 group than the control and other groups. In subsequent trials, the expression of the FGF2 transcript was higher (P < 0.05) in A-grade of oocytes than the C- and D-grade of oocytes, but the expression was not different (P> 0.05) from the B-grade of oocytes. While the FGFR2 expression was higher (P < 0.05) in cumulus cells than any grades of oocytes. The relative abundance of FGF2 and FGFR2 transcripts was significantly higher (P < 0.05) in the 2-cell stage of the embryo than the other stages of embryos. This study was further extended to characterize the FGF2 ligand-binding site in the D3 domain of the buffalo FGF2 receptor. Bioinformatics analysis showed that the bovine FGF2 ligand-binding site in the D3 domain of buffalo was different from the D3 domain of the cattle.

Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions.

In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.

RNA sequencing and transcriptome analysis of buffalo (Bubalus bubalis) blastocysts produced by somatic cell nuclear transfer and in vitro fertilization.

Across farm animal species, the live birth rate obtained with somatic cell nuclear transfer (SCNT) embryos is only <2% compared with >40% obtained with in vitro fertilization (IVF) embryos, primarily due to incomplete nuclear reprogramming which results in aberrant embryonic gene expression. We used RNA sequencing to compare the global transcriptome profile of SCNT and IVF buffalo blastocysts. SCNT blastocysts expressed 17,061 transcripts, of which 941 were unique whereas, IVF blastocysts expressed 17,303 transcripts, of which 1,183 were unique. At ≥2-folds change (p < .05), 331 transcripts were differentially expressed in the two groups among which, 19 were unique, 188 were downregulated and 143 were upregulated in SCNT compared with IVF blastocysts. Many genes affecting pluripotency, trophectoderm development, developmental regulation, and epigenetic modifications were upregulated in SCNT compared with IVF blastocysts. Among the four functional categories analyzed, epigenetic regulators were the most affected. Most of the WNT signaling pathway genes were upregulated whereas, the inhibitors of this pathway, such as DKK1, were downregulated in SCNT blastocysts, suggesting that this pathway is overexpressed in SCNT embryos. Gene Ontology analysis revealed that 25 biological processes, 20 molecular functions, and 24 cellular compartment categories were enriched in SCNT blastocysts. This data can help identify reprogramming errors for improving cloning efficiency.

Selection of Reference miRNAs for Relative Quantification in Buffalo (Bubalus bubalis) Blastocysts Produced by Hand-Made Cloning and In Vitro Fertilization.

Very low birth rate and a high incidence of abnormalities in offspring born from cloned embryos, which have limited the application of cloning technology on a wide scale, are believed to be because of incomplete or aberrant nuclear reprogramming. MicroRNAs (miRNAs) are involved in regulating a wide range of biological processes including reprogramming and embryonic development. Selection of suitable reference miRNAs is critical for normalization of data for accurate relative quantification of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR), which is currently the most widely used technique for quantifying miRNAs. This study was aimed at identification of reference miRNAs suitable for normalization of qRT-PCR data from blastocyst-stage buffalo embryos produced by handmade cloning and in vitro fertilization (IVF). RNA isolated from cloned and IVF blastocysts was subjected to next-generation sequencing based on which, 12 highly and most consistently expressed miRNAs, which included miR-92a, miR-423, miR-151, Let-7a, miR-103a, miR-93, miR-16b, miR-25, miR-30e, miR-101, miR-127, and miR-197, were selected as candidates for identification of suitable reference miRNAs using three statistical algorithms namely geNorm, NormFinder, and BestKeeper. Based on consensus of the three algorithms, the combination of miRNAs found to be suitable as reference miRNAs were miR-127 and miR-103 for IVF blastocysts; miR-92a and miR-103 for cloned blastocysts, and miR-103, miR-423, and miR-93 across both IVF and cloned blastocysts. The data of this study can be very useful in miRNA expression analysis of blastocyst-stage cloned and IVF embryos.

Treatment of Buffalo (Bubalus bubalis) Somatic Cell Nuclear Transfer Embryos with MicroRNA-29b Mimic Improves Their Quality, Reduces DNA Methylation, and Changes Gene Expression Without Affecting Their Developmental Competence.

microRNA-29b (miR-29b) plays an important role in controlling DNA methylation in cells. We investigated its role during early embryonic development in buffalo embryos produced by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). miR-29b expression was highest at the 2-cell stage, decreased (p < 0.001) at the 4-cell stage, and remained low thereafter at the 8-cell, morula, and blastocyst stages, showing a similar pattern in cloned and IVF embryos. Treatment of reconstructed embryos with miR-29b mimic for 1 hour after 1 hour of electrofusion increased (p < 0.05) the total cell number and decreased (p < 0.05) the levels of apoptosis and DNA methylation compared with controls. It also increased (p < 0.05) the ratio of inner cell mass:trophectoderm cell numbers of blastocysts compared with controls to the levels observed in IVF blastocysts. However, the blastocyst rate was not affected by treatment with miR-29b mimic (29.0% ± 2.0% vs. 27.0% ± 2.0% for controls). The treatment decreased (p < 0.001) the expression of epigenetic-related genes, DNMT3A and DNMT3B, but not DNMT1, and increased (p < 0.05) that of pluripotency- (NANOG, OCT4, and SOX2) and development-related genes (FGF4 and GLUT1) in blastocysts compared with controls. Our results suggest that miR-29b mimic treatment of reconstructed embryos improves the quality, reduces the level of apoptosis and DNA methylation, and changes gene expression in SCNT blastocysts without affecting the blastocyst rate.

Optimization of Serum-Free Culture Conditions for Propagation of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) self-renew and produce a large number of differentiated germ cells to maintain normal spermatogenesis. However, the growth factors crucial for SSC self-renewal and the mechanism underlying this process remain unclear. In the present study, a serum-free culture media was used to evaluate the effect of several growth factors on the expression of some SSC markers and self-renewal related genes. The putative SSCs were cultured on buffalo Sertoli cell feeder layer in KO-DMEM +10% KOSR. The colony formation was observed between 7 and 10 days. The putative SSC colonies also expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days, relative mRNA expression study revealed that 20 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of PLZF, TAF4B, and THY1. Furthermore, supplementation of a combination of 20 ng/mL GDNF, 10 ng/mL basic fibroblast growth factor (bFGF), 1000 IU/mL leukemia inhibitory factor (LIF), and 1 ng/mL colony stimulating factor 1 (CSF1) upregulated the expression of PLZF, TAF4B, BCL6B, and ID4 genes. These results demonstrated that our defined culture media in combination with GDNF, bFGF, LIF, and CSF1 well supported SSC self-renewal.

Supplementation of Glial Cell Line-Derived Neurotrophic Factor, Fibroblast Growth Factor 2, and Epidermal Growth Factor Promotes Self-Renewal of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells by Upregulating the Expression of miR-20b, miR-21, and miR-106a.

In this study, we investigated the effect of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2, and epidermal growth factor (EGF) on the expression of some self-renewal-related microRNAs (miRs) in putative buffalo spermatogonial stem cells (SSCs). The SSCs were cultured on a buffalo Sertoli cell feeder layer, colony formation was observed between 7 and 10 days. The SSC colonies expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days of initial culture, the colonies were subcultured as treatment (supplemented with 20 ng mL-1 GDNF +10 ng mL-1 FGF2 + 10 ng mL-1 EGF) and control groups. The number and area of SSC colonies were significantly (p < 0.05) higher in the treatment group than in the control group. The relative abundance of miR-20b, miR-21, and miR-106a in SSCs supplemented with growth factors was significantly higher (p < 0.001) than that in the control. The results indicate that supplementation of SSC culture medium with growth factors (GDNF, FGF2, and EGF) may promote the expression of miR-20b, miR-21, and miR-106a, which is essential for self-renewal and maintenance of SSCs.

An update: Reproductive handmade cloning of water buffalo (Bubalus bubalis).

The first birth of a cloned animal produced through the Handmade cloning (HMC) technique was reported more than 15 years ago in cattle. This method of somatic cell nuclear transfer (SCNT) has subsequently been evolving as a much simpler alternative to the classical micromanipulator-based SCNT. Several farm animal species such as cattle, buffalo, pigs, sheep, and goats have been successfully cloned using HMC. In buffalo, HMC technique is now well established, and several births of cloned calves have been reported by us. Several factors such as source of somatic cells, quality of recipient oocytes, cell cycle stage prior to SCNT, electrofusion and culture conditions, and epigenetic status of somatic cells, have been optimized leading to the production of good quality cloned embryos. The preservation through cloning of proven breeding bulls that have died by producing live offspring using somatic cells isolated from frozen semen as donor cells and birth of a cloned calf from urine-derived cells are impressive examples of the success of HMC in buffalo. In conclusion, HMC is a valued reproductive technique in buffalo that offers the opportunity to make multiple copies of highly valuable animals, particularly proven breeding bulls. In this review, there is a discussion of the advancement of the HMC technique in buffalo and factors responsible for the efficient production of cloned embryos.

Buffalo SCNT embryos exhibit abnormal gene expression of ERK/MAPK pathway and DNA methylation.

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down-regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real-time PCR in buffalo blastocysts produced by Hand-made cloning and compared it with that in blastocyst-stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.e., p21RAS, RAF1, AKT1, ERK2, PIK3R2 and c-Myc was significantly higher (p < 0.05) in cloned than in IVF embryos. However, the expression level of FOS was lower (p < 0.005) in cloned than in IVF embryos. The relative expression level of DNMT3a and DNMT3b but not that of DNMT1 was significantly higher (p < 0.05) in cloned than in IVF embryos. These results indicate that the cloned embryos exhibit an abnormal expression of several important genes related to ERK/MAPK pathway and DNMTs. Although a direct link between ERK/MAPK pathway and DNMTs was not examined in the present study, it can be speculated that ERK/MAPK pathway may have a role in regulating the expression of DNMTs in embryos, as also observed in other tissues.

Cloning of Buffalo, a Highly Valued Livestock Species of South and Southeast Asia: Any Achievements?

Buffalo (Bubalus bubalis) is a major source of milk, meat, and draught power in many developing countries in Asia. Animal cloning holds a lot of potential for fast multiplication of elite buffaloes and conservation of their valuable germplasm. Although the progress of buffalo cloning has been slow in comparison to cattle or pig, several breakthroughs were reported in buffalo cloning such as the production of cloned calves from somatic cells isolated from over one-decade old frozen-thawed semen or from urine-derived cells. Since the initiation of buffalo cloning, several approaches have been tried to refine nuclear transfer protocols. This has resulted in increasing the blastocyst production rate and improving their quality leading to an increase in live birth rate. In this review, we discuss current developments in buffalo cloning, its challenges, and the future roadmap.

Establishment, Growth, Proliferation, and Gene Expression of Buffalo (Bubalus bubalis) Transgenic Fetal Fibroblasts Containing Human Insulin Gene, and Production of Embryos by Handmade Cloning Using These Cells.

The aim of the present study was to compare transgenic cells, containing human insulin gene kept under the control of mammary gland-specific buffalo beta-lactoglobulin promoter, and their counterparts, that is, nontransgenic cells, for examining their potential for the production of embryos following somatic cell nuclear transfer (SCNT). The gene construct was delivered into buffalo fetal fibroblasts (BFF) by nucleofection following which, the transfected cells were selected by culture in the presence of G418 for 3 weeks. Transgene integration into BFF genome was confirmed by polymerase chain reaction (PCR) and reverse transcriptase PCR. At passage 8-10, the growth rate, cell proliferation rate, and quantitative expression of certain genes were compared between transgenic and nontransgenic cells. The growth rate and cell proliferation rate was significantly lower (p < 0.05) for transgenic than for nontransgenic cells. Using quantitative real-time PCR it was found that the expression level of CASPASE 3, CASPASE 9, BAX, and P53 was significantly higher (p < 0.05) and that of HDAC1 and IGF-1R was significantly lower (p < 0.05) in transgenic compared with nontransgenic cells. The differences in the relative expression level of BCL-XL, MCL-1, DNMT1, DNMT3a, GDF9, FGF2, and G6PD between the two groups were not significant. Furthermore, when the two cell types were used as donor cells for production of embryos by handmade cloning, the blastocyst rate was significantly lower (p < 0.05) with transgenic (35.69% ± 1.78%) than with nontransgenic cells (48.75% ± 2.38%). In conclusion, these results indicate that differences were present between transgenic and nontransgenic cells, which may affect the efficiency of SCNT when used as donor cells.