Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning
The current study aimed to explore the effects of miR- 449b on the epigenetic status of transgenic embryos (contain- ing human insulin gene) produced through handmade cloning. For this, in vitro matured oocytes were enucleated by micro- blade, and two demi-oocytes were fused with a somatic cell (tr...
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| Main Author: | |
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| Format: | Article |
| Language: | English |
| Published: |
Universidad del Zulia
2023-11-01
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| Series: | Revista Científica |
| Subjects: | |
| Online Access: | https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43499 |
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| Summary: | The current study aimed to explore the effects of miR-
449b on the epigenetic status of transgenic embryos (contain-
ing human insulin gene) produced through handmade cloning.
For this, in vitro matured oocytes were enucleated by micro-
blade, 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) fluo-
rescence dye] and the effect of miR-449b on epigenetic status
was studied by immunofluorescence and qPCR. The transgen-
ic 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 pro-
duced 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 reconstruct-
ed embryos were cultured for 2 h with miR-449b mimic as com-
pared 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 sig-
nificantly higher (p<0.05) up to 4.89±0.39 fold compared to the
control. However, in the case of DNMT1, there was no signif-
icant 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.
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| ISSN: | 0798-2259 2521-9715 |