ABSTRACT
Background: nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression
Objective: the impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study
Materials and Methods: in this experimental study, 8 cell embryos [n=240] were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh [n=80], vitrified at 8 cell stage [n=80], vitrified at 8 cell stage thawed and re-vitrified at compaction stage [n=80]. Embryos were vitrified by using cryolock, [open system] described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts
Results: our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos [p=0.03]. In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos [p=0.004], however expression of Bax and Bcl-2 [apoptotic] genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos [p=0.003], but it was similar between re-vitrified and vitrified embryos
Conclusion: re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage
ABSTRACT
Objective: Vitrification is a convenient, effective method for freezing and storing embryos. Under certain situations, such as an unsuitable endometrial environment, extra embryos can be re-vitrified for future use. There is inadequate data on the effects of revitrification on embryos, so we have evaluated the effects of re-vitrification on the development rate and expression of apoptotic and implantation genes
Methods: Female NMRI mice, ages six-eight weeks were super-ovulated with 7.5 IU PMSG and 7.5 IU hCG. Females were mated with males from the same strain and inspected for the presence of vaginal plugs the following morning. Females with the presence of vaginal plugs were considered to be pregnant and killed 62 h post hCG injection. Eight-cell embryos were flushed from their oviducts and subsequently divided into three experimental groups: fresh, vitrified-warmed 8-cell embryos, and re-vitrifiedwarmed blastocyst embryos. RNA was extracted and we used real-time PCR to evaluate expressions of Bax, Bcl-2, and ErbB4. Data was analyzed by the chi-square and ANOVA tests
Results: A significant difference existed in blastocyst formation rate, degeneration rate, and expressions of Bax, Bcl-2, and ErbB4 in re-vitrified embryos compared to fresh embryos
Conclusion: The vitrification and warming process did not affect the developmental rate and expressions of Bax, Bcl-2, and ErbB4 in the eight-cell stage embryos. However, we observed a change in development rate and expression rates of Bax, Bcl-2, and ErbB4 after re-vitrification in the early blastocyst stage
ABSTRACT
Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site [IRES] sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics