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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Universidad del Zulia
2023-11-01
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| Online Access: | https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43499 |
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| author | Gaurav Tripathi |
| author_facet | Gaurav Tripathi |
| author_sort | Gaurav Tripathi |
| collection | DOAJ |
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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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| format | Article |
| id | doaj-art-3054afce62ef4246a54127856b270421 |
| institution | Kabale University |
| issn | 0798-2259 2521-9715 |
| language | English |
| publishDate | 2023-11-01 |
| publisher | Universidad del Zulia |
| record_format | Article |
| series | Revista Científica |
| spelling | doaj-art-3054afce62ef4246a54127856b2704212025-02-06T15:37:05ZengUniversidad del ZuliaRevista Científica0798-22592521-97152023-11-0133SuplementoMir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloningGaurav Tripathi0Embryo Biotechnology Lab, Animal Biotechnology Division ICAR- National Dairy Research Institute, Karnal-132001, India 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. https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43499buffalomiR-449bhandmade cloningembryoepigenetics |
| spellingShingle | Gaurav Tripathi Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning Revista Científica buffalo miR-449b handmade cloning embryo epigenetics |
| title | Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning |
| title_full | Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning |
| title_fullStr | Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning |
| title_full_unstemmed | Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning |
| title_short | Mir-449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning |
| title_sort | mir 449b improves the epigenetic status of buffalo transgenic embryos produced by handmade cloning |
| topic | buffalo miR-449b handmade cloning embryo epigenetics |
| url | https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43499 |
| work_keys_str_mv | AT gauravtripathi mir449bimprovestheepigeneticstatusofbuffalotransgenicembryosproducedbyhandmadecloning |