Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer

2009 ◽  
Vol 21 (6) ◽  
pp. 785 ◽  
Author(s):  
Angelica M. Giraldo ◽  
John W. Lynn ◽  
Megan N. Purpera ◽  
Todd D. Vaught ◽  
David L. Ayares ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Expression Patterns ◽  
Subsequent Development ◽  
Nuclear Transplantation ◽  
Fibroblast Cells ◽  
Dna Methyltransferase 1 ◽  
Donor Genome ◽  
Aberrant Expression ◽  
Methyltransferase 1

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the ΔΔCT method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.

30 INHIBITION OF DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE FIBROBLAST CELLS FOR NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
A. M. Giraldo ◽  
J. W. Lynn ◽  
M. N. Purpera ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Transfection Efficiency ◽  
Expression Patterns ◽  
Preimplantation Embryos ◽  
Dna Methyltransferase 1 ◽  
Aberrant Expression ◽  
Transfection Reagent ◽  
Fetal Fibroblasts ◽  
Methyltransferase 1

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer (NT). DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. NT utilizing cell fusion introduces the somatic form of DNMT1 (DNMT1s), which is not normally present in preimplantation embryos and could perpetuate the somatic-like methylation patterns observed in early cloned embryos. The objective of this study was to decrease the level of DNMT1s in bovine fibroblasts using siRNA, prior to their use as donor cells. Fetal fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2 at 37�C. The transfection efficiency of different ratios of siRNA concentrations and transfection reagent (µg:µL; 1:3, 1:6, and 1:9) as well as siRNA concentrations (0.5, 1.0, and 1.5 µg) were determined using fluorescein isothiocyanate (FITC)-labeled control siRNA and fluorescence analyzed by flow cytometry. A DNMT1s-specific siRNA was used to transfect cells at 50 and 80% of confluence. A non-silencing siRNA was used as a negative control. The expression patterns of DNMT1s were characterized by Q-PCR using the δδCT method. ANOVA was used to detect differences in transfection efficiency and gene expression. The combination of 1.0 or 1.5 µg siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Cells treated at 50% confluence with 1.5 µg of DNMT1s-specific siRNA at a 1:6 ratio showed 80% less DNMT1s mRNA than cells treated with non-silencing siRNA. At 8 h post-transfection (PT) these cells displayed vacuole-like structures within the cytoplasm, stopped dividing, and died approximately 24 h PT. Cells treated at 80% confluence with 1.0 µg DNMT1s-specific siRNA at a 1:6 ratio resulted in 57, 66, 24, 50, 22, 62, 64, and 56% less DNMT1s than control cells at 4, 6, 8, 10, 12, 24, 48, and 72 h PT, respectively, but without the cytotoxic effects observed when cells at 50% of confluence were treated under the same transfection conditions. These data indicate that optimization of cell density, siRNA concentration, and the ratio between siRNA concentration and transfection reagent are required to decrease the levels of DNMT1s without causing deleterious effects to the cells. However, levels of DNMT1s mRNA can be effectively reduced using siRNA; protein analysis is required to determine if reduction of the transcript results in lower levels of the protein. Subsequent use of these cells for NT will provide insight as to how the presence of this enzyme affects reprogramming in early cloned embryos. This study was supported by Louisiana State University Board of Regents.

45 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE PARTHENOTES AND NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
R. D. W. Kelly ◽  
R. Alberio ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Expression Patterns ◽  
Subsequent Development ◽  
Lamin A ◽  
Santa Cruz ◽  
Cell Stage ◽  
Aberrant Expression ◽  
Fetal Fibroblasts ◽  
Bovine Oocytes

Despite the apparent successes of nuclear transfer (NT) technology, numerous recent reports have indicated de-regulation of key gene expression patterns in NT embryos as compared to their in vivo and IVF counterparts. Aberrant expression of lamin A/C has been reported in mouse (Moreira et al. 2003 J. Cell Sci. 116, 3713-3720) and bovine (Sullivan et al. 2004 Biol. Rep. 70, 146-153) NT embryos, leading to the hypothesis that the presence of lamin A/C might affect subsequent development. Lamin A/C expression is a potential marker for reprogramming due to the induced expression and remodeling during differentiation. Previously using immunofluorescence in bovine IVF embryos, we have demonstrated the persistence of lamin A/C until the 2-cell stage (Kelly et al. 2005 Reprod. Fertil. Dev. 17, 205-206). The present study was initiated to further characterize lamin A/C expression in bovine parthenogenetic and NT embryos using a monoclonal antibody specific to lamin A/C. Bovine oocytes were matured in vitro as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255-262). NT embryos were constructed using lamin A/C-positive primary bovine fetal fibroblasts and in vitro-matured, enucleated MII bovine oocytes. Oocyte cell couplets were fused at 24 h post onset of maturation 1 h prior to activation. Oocyte activation was achieved with 7% ethanol for 7 min followed by a 6 h incubation in mSOF containing 10 �g/mL cycloheximide and 7.5 �g/mL cytochalasin B for the production of both NT and parthenogenetic embryos. Embryos were cultured in mSOF supplemented with 10% FCS and collected at various stages for immunofluorescence staining. Prior to fixation, embryos were incubated in 2 mg/mL protease to remove the zona pellucida. Samples were fixed in 100% methanol at -20�C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Embryos were then incubated overnight at 4�C with mouse anti-lamin A/C antibody (IgM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with Cy3 goat anti-mouse IgM (1:400) (Chemicon International, Inc., Temecula, CA, USA) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were mounted in VectaShield mounting medium containing 42,6-diamidino-1-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were viewed using epifluorescence (Leica DMR; Leica Microsystems, Wetzlar, Germany) and confocal microscopy (Leica TCS). Inhibiting protein synthesis during the activation period with cycloheximide had no effect on lamin A/C assembly in 6 h post activation (hpa) parthenogenetic (35/35) and NT (7/7) embryos. The pronuclei of parthenogenetic (30/30) and NT (15/15) zygotes at 22 hpa were also positively labeled for lamin A/C. Nuclear labeling was observed in both parthenogenetic (25/25) and NT (12/12) 2-cell embryos. All parthenogenetic and NT embryos examined from the 4-cell stage through to blastocysts were stained negatively for lamin A/C. These results suggest that lamin A/C present in bovine NT zygotes is not due to aberrant reprogramming and that remodeling of the nuclear lamina occurs correctly in bovine NT embryos.

56 EFFECTS OF KNOCKDOWN OF DNA METHYLTRANSFERASE 1 BY RNA INTERFERENCE ON IN VITRO DEVELOPMENT AND DNA METHYLATION STATE IN BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 128
Author(s):  
K. Yamanaka ◽  
M. Sakatani ◽  
M. Takahashi
Keyword(s):  
Dna Methylation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Epigenetic Modifications ◽  
Dna Methyltransferase 1 ◽  
In Vitro Development ◽  
Vitro Development ◽  
Methyltransferase 1

Reprogramming of epigenetic modification is a necessary process during mammalian development, which is aberrant in somatic cell nuclear transfer (SCNT) embryos. Previous study has demonstrated that an abnormal state of genomic hypermethylation is consistently observed in SCNT embryos (Kang et al. 2001 Nat. Genet. 28, 173–177). On the other hand, small interfering RNAs (siRNAs) are identified molecules shown to silence genes via targeted mRNA degradation and are widely used in molecular and cellar research (Hannon GJ 2002 Nature 418, 244–251). Thus, knockdown of the expression of genes related to epigenetic modifications by siRNA may be used to alter epigenetic modifications in SCNT embryos. In the present study, we investigated the effects of knockdown of DNA methyltransferase 1 by siRNA on in vitro development, gene expression, and DNA methylation state of bovine SCNT embryos. In vitro matured oocytes were enucleated, fused with bovine fibroblasts and then activated, the resultant SCNT embryos were divided into three groups; control, non-treated group; sham-NT, H2O injected group; and siRNA-NT, siRNA injected group. The siRNA corresponding to DNA methyltransferases 1, which is the enzyme responsible for maintaining DNA methylation patterns, was designed and injected into the cytoplasm of SCNT embryos. All embryos were cultured in CR1aa + 5% FCS and assessed the rates of cleavage and blastocyst formation on Days 2 and 8, respectively. All data were obtained from more than 5 replicates. Developmental percentage data were analyzed by chi-square tests (P < 0.05). Other data were analyzed with ANOVA followed by Fisher’s protected least significant difference (P < 0.05). The developmental rate to blastcysts in siRNA-NT group (38.7%; 111/287) was significantly higher (P < 0.05) than those of control (28.8%; 121/420) and sham groups (30.5%; 92/302). To estimate the effect of siRNA injection on gene expression, we sampled embryos at 48 h after culture and measured the amount of DNA methyltransferase 1 mRNA expression by real-time PCR. The amount of DNA methyltransferase 1 mRNA was significantly less (P < 0.05) than those of control and sham-NT groups. Finally, the levels of DNA methylation at satellite I region were analyzed by COBRA method in blastosyst stage embryos. The level of DNA methylation of blastocysts in siRNA-NT groups was significantly less (P < 0.05) than those of control and sham-NT and also similar to that of IVF blastocysts. In the present study, we showed that gene silencing of DNA methyltransferase 1 by siRNA enhanced the in vitro development of SCNT embryos and decreased the level of DNA methylation which was equivalent to IVF embryos. These findings suggest that knockdown of specific genes related epigenetic modifications by RNA interference may alter abnormal epigenetic reprogramming with the resultant improvement for subsequent development of SCNT embryos.

Folate Deficiency and Aberrant Expression of DNA Methyltransferase 1 were Associated with Cervical Cancerization

2014 ◽  
Vol 20 (11) ◽  
pp. 1639-1646 ◽  
Author(s):  
Wang Jin-tao ◽  
Ding Ling ◽  
Jiang Shi-Wen ◽  
Hao Junxia ◽  
Zhao Wei-min ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Folate Deficiency ◽  
Dna Methyltransferase 1 ◽  
Aberrant Expression ◽  
Methyltransferase 1
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