Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning

• Gaurav Tripathi Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India
• Sonal Gupta Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India
• Ram Parsad Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India
• Tanya Gupta Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India
• Naresh L. Selokar Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India
• Manoj Kumar Singh Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India

DOI: https://doi.org/10.52973/rcfcv-wbc126

Keywords: buffalo, miR-449b, handmade cloning, embryo, epigenetics

Abstract

The current study aimed to explore the effects of miR449b on the epigenetic status of transgenic embryos (containing human insulin gene) produced through handmade cloning. For this, in vitro matured oocytes were enucleated by microblade, and two demi-oocytes were fused with a somatic cell (transgenic cells) by electrofusion and after 1 h of electrofusion, the fused embryos (reconstructs) were incubated in 40 nM miR- 449b mimic/inhibitor by lipofection method for 1 h at 38.5°C in a CO2 incubator. After incubation, transfected zygotes were chemically activated and subjected to in vitro culture in RVCL (Cook®) medium for 8 days at 38.5°C in 5% CO2 in air, 90-95% relative humidity condition. Transfection of mi-449b was confirmed through the red fluorescence produced by miR-449b [tagged with 5 carboxytetramethylrhodamine (TAMARA) fluorescence dye] and the effect of miR-449b on epigenetic status was studied by immunofluorescence and qPCR. The transgenic embryos showed a similar development pattern as control embryos. The global level of 5-methyl cytosine was compared among three groups of buffalo transgenic cloned embryos produced by treatment of reconstructed embryos with miR-449b control, mimic, and inhibitor. It was found that the mean pixel intensity was significantly reduced (p<0.05) when reconstructed embryos were cultured for 2 h with miR-449b mimic as compared to control (mimic 7.92 ± 0.34 vs control 16.15 ± 0.50). At the same time, it was not significantly higher (p>0.05) when reconstructed embryos were exposed to miR-449b inhibitor as compared to control (Inhibitor 17.18 ± 0.88 vs control 16.15 ± 0.50). The relative expression level of HDAC1 and DNMT1 were significantly reduced (p<0.05), up to 0.33±0.39 fold and 0.16±0.74 fold, respectively, compared to control when treated with miR-449b mimic. At the same time, in the case of miR-449b inhibitor, the expression level of HDAC 1 gene was significantly higher (p<0.05) up to 4.89±0.39 fold compared to the control. However, in the case of DNMT1, there was no significant difference (p<0.05) 1.17±0.66 fold compared to control embryos. This study revealed that miR-449b mimic reduces the global methylation level of buffalo transgenic cloned embryos, hence improving developmental competence and quality.