title: Downregulation of DNA methyltransferase 1 in zona-free cloned buffalo (Bubalus bubalis) embryos by small interefering RNA improves in vitro development but does not alter DNA methylation level.
abstract: Aberrant epigenetic reprogramming, especially genomic hypermethylation, is implicated as the primary reason behind the failure of the cloning process during somatic cell nuclear transfer (SCNT). We transfected one-cell-stage zona-free buffalo embryos produced by handmade cloning with 50 nM DNMT1 small interfering RNA (siRNA), using lipofectamine, to knockdown the DNA methyltransferase 1 (DNMT1) gene. siRNA treatment decreased (p<0.001) the expression level of DNMT1 mRNA and DNMT1 protein in the one-cell-stage embryos and increased (p<0.05) the blastocyst rate (52.3 ± 1.3% vs. 45.3 ± 2.5%) compared to that in the controls, but did not reduce the DNA methylation level similar to the in vitro-fertilized (IVF) embryos. It also increased (p<0.05) the relative mRNA abundance of P53 and CASPASE 3, but not that of HDAC1, DNMT1, and DNMT3a, in the blastocysts of the siRNA group compared to the controls. The global level of H3K18ac was higher (p<0.05) in the blastocysts of the siRNA group than in the controls, whereas that of H3K9ac and H3K27me3 was not significantly different between the two groups. In conclusion, lipofection can be successfully used for transfection of DNMT1 siRNA into one-cell-stage zona-free cloned buffalo embryos. It results in a concomitant decrease in the DNMT1 mRNA and protein levels in the one-cell-stage embryos. siRNA-mediated knockdown increases the blastocyst rate but does not alter the DNA methylation level.
authors.affiliation: 1 Animal Biotechnology Centre, National Dairy Research Institute , 132001 Karnal, India .
authors.forename: Naresh L
authors.initials: NL
authors.lastname: Selokar
authors.forename: Monika
authors.initials: M
authors.lastname: Saini
authors.forename: Himanshu
authors.initials: H
authors.lastname: Agrawal
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
authors.forename: Radheysham
authors.initials: R
authors.lastname: Manik
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D002020:Buffaloes / Q000235:genetics*; D019976:Cloning, Organism / Q000379:methods*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation; D015536:Down-Regulation; D047108:Embryonic Development; D018507:Gene Expression Regulation, Developmental; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004248:DNA (Cytosine-5-)-Methyltransferases
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