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- Title
Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning.
- Authors
Tripathi, Gaurav; Gupta, Sonal; Parsad, Ram; Gupta, Tanya; Selokar, Naresh L.; Singh, Manoj Kumar
- Abstract
The current study aimed to explore the effects of miR-449b 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.
- Subjects
WATER buffalo; EPIGENETICS; ANIMAL cloning
- Publication
Revista Cientifica de la Facultade de Veterinaria, 2023, Vol 33, p282
- ISSN
0798-2259
- Publication type
Article
- DOI
10.52973/rcfcv-wbc126