Cloning and characterization of buffalo NANOG gene: alternative transcription start sites, splicing, and polyadenylation in embryonic stem cell-like cells.

NANOG is a critical homeodomain transcription factor responsible for maintaining embryonic stem cell (ESC) self-renewal and pluripotency. In the present study, we isolated, sequenced, and characterized the NANOG gene in buffalo ESC-like cells. Here, we demonstrated that NANOG mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and five different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 182, 95, 35, and 17 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which leads to the expression of at least five different 3'-untranslated regions (269, 314, 560, 566, and 829 nucleotides). Expression analysis showed that these alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open reading frame of buffalo NANOG codes for a 300-amino-acid-long protein. Further, results showed that alternative splicing leads to the expression of two types of transcript variants encoded by four and five exons. In silico analysis of cloned 5'-flanking region (3366 nucleotides upstream of translation start codon) identified several putative transcription factors binding sites in addition to a TATA box and CAAT box at -30 and -139 bp (upstream to the distal most TSS), respectively, in the buffalo NANOG promoter.

Characterization of POU5F1 (OCT4) gene and its promoter in buffalo ESC-like cells identifies multiple transcription start sites and expression of four pseudogenes.

In the present study, we cloned and characterized the buffalo (Bubalus bubalis) OCT4 ortholog expressed in embryonic stem cell (ESC) like cells and its promoter region. The 5'- and 3'-RACE experiments were conducted to analyze the transcription initiation site and regulatory regions. The comparative analysis of buffalo OCT4 promoter with other mammalian orthologs revealed high conservation. Among the regulatory regions highest similarity was observed between buffalo, bovine and sheep. Interestingly, buffalo OCT4 promoter exhibited a 78 bp deletion between two proximal enhancers (PE-1A and PE-1B) when compared to other mammalian orthologs. 5'-RACE revealed four different transcription start sites for OCT4 gene. As far as we know there is no previous report regarding multiple transcription initiation sites for OCT4 gene in any species. In addition, we identified expression of four pseudogenes in buffalo ESC-like cells. Among the multiple transcripts characterized, we found four cDNA clones (1083 bp) derived from ESC-like cells sharing 96.9-99.3% sequence homology with the parent gene and having the capacity of encoding 139, 206, 206 and 324 amino acid long truncated proteins. Multiple pseudogenes have been proposed for OCT4 which might contribute to the false detection of this gene during expression studies. However, only few of them were reported to be transcribed and none were reported to be translated in stem cells. Western blot analysis of OCT4 protein using ESC-like cells revealed multiple bands, indicating that some of the hypothetical pseudogenes are being translated. These novel pseudogenes or their protein products may have some important regulatory functions.

Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.