3 research outputs found
Study of factor XI deficiency in Khuzestan cattle population of Iran
The present study investigated the occurrence of autosomal recessive genetic disease, factor XI (FXI), in Khuzestan native cows and Iranian Holstein cattle. Genomic DNA was isolated from the blood of the cows (n = 330). Exon 12 of the Factor XI gene of the cows was amplified by polymerase chain reaction (PCR). Additionally, all cows were confirmed by DNA sequencing to determine existence of mutant FXI allele. Normal cattle have only one DNA fragment of 244 bp while heterozygous cattle exhibited two DNA fragments of 320 and 244 bp for the FXI gene deficiency. The results of this study showed that none of the animals were carriers of FXI deficiency. Because of the economical significance of the FXI mutation and its recessive mode of inheritance, attention has to be paid to any case of a bull having in his origin any known FXI carrier. Although we did not observe any carrier, widespread screening programs for detection of genetic disorders seems necessary.Key words: Factor XI, deficiency, sequencing, cattle, Iran
Optimization of in vitro culture and transfection condition of bovine primary spermatogonial stem cells
The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase (AP) staining and expression of vasa and thy1 genes as specific bovine SSC markers. To evaluate the effect of antioxidants on vitality, colony formation, and the expression of pro- and anti-apoptotic genes of bovine SSCs, various concentrations of vitamin C (5, 10, 25 and 50 μg/mL) and Trolox (a water soluble α-tocopherol analogue) (12.5, 25, 50 and 100 μg/mL) were added to the SSC culture medium. The results showed that SSCs treated with 50 μg/mL of vitamin C or 25 μg/mL of Trolox individually could increase cell viability and colony formation significantly in comparison with other concentrations and the control group. Additionally, the expressions of bax (as a pro-apoptotic gene) and bcl2 (as an anti-apoptotic gene) were significantly lower and higher than the control group, respectively. To optimize the transfection condition, the effective dosages of vitamin C or Trolox, with various concentrations of two transfection reagents (X-tremeGENE HP and Turbofect) and DNA, at day 8 of culture, were studied. Results showed that 1 μl X-tremeGENE HP or 0.5 μl Turbofect and 2 μg of DNA are the best concentrations for transfecting SSCs. However, X-tremeGENE HP expressed more potential for transfecting SSCs in comparison with Turbofect. Besides, no difference was observed between the use of defined doses of vitamin C or Trolox.Keywords: Apoptosis, gene transfer, primary cells, viability, vitamin C, Trolo
Evaluating bovine sperm transfection using a high-performance polymer reagent and assessing the fertilizing capacity of transfected spermatozoa using an in vitro fertilization technique
Sperm-mediated gene transfer (SMGT) has been considered as an innovative
device for transgenesis on a mass scale by taking advantage of live
spermatozoa to transfer exogenous DNA. However, the fertilizing ability of
transfected sperm cells and the poor reproducibility of this method are still
matters of controversy. Hence, the current study was conducted to evaluate
transfecting the enhanced green fluorescent protein (EGFP) as the source of
exogenous DNA into bovine spermatozoa using a high-performance polymer
reagent as well as assessing the fertilizing capacity of transfected sperm
cells by in vitro fertilization (IVF). In the first experiment, three
different concentrations of rhodamine-labeled DNA and high-performance
polymer transfection reagent, X-tremeGENE HP, were used to transfect bovine
spermatozoa. In the second experiment, IVF and fluorescence microscopy
methods were utilized to assess the fertilizing capacity of sperm cells
carrying exogenous DNA when X-tremeGENE HP was used either alone or with
dimethyl sulfoxide (DMSO) treatment. Findings revealed that at 1 µL
X-tremeGENE HP and 1 µg of DNA concentration, approximately
one-third of total spermatozoa were transfected. However, following IVF and
fluorescence microscopy, no EGFP expression was detected in zygotes and
morula-stage embryos. Results of this study showed that, although X-tremeGENE
HP could transfer EGFP to bovine spermatozoa, transfected sperm cells were
unable to transfer foreign DNA to matured bovine oocytes. Under our
experimental conditions, we hypothesized that the absence of the EGFP
fluorescence signal in embryos could be due to the detrimental effects of
transfection treatments on sperm cells' fertility performance as well as
incompetency of IVF to produce transgenic embryos using transfected sperm
cells.</p