We report a method for gene transfer via in vivo testis-mediated gene transfer (TMGT) in sheep. A non-viral vector, pDB2, which carried an enhanced green fluorescent protein (EGFP) transgene under control of a human cytomegalovirus (CMV) promoter, was mixed with the TransIT transfection reagent. The lipoplex mixture was injected intra-testicularly or into the cauda epididymis of 12 and 5 rams, respectively, as well as the intra-testis of 5 mature roosters. Each injected rooster was crossed with four virgin hens and their hatched chicks were assessed for the presence of the EGFP transgene. After 60 days, both rams and roosters were investigated for the transgene. Unlike roosters, polymerase chain reaction (PCR) analysis of sperm cells which were collected from the epididymis and seminiferous revealed that more than 50% of sperm samples from the intratesticular rete-injected group were PCR-positive for the EGFP transgene. Transgene uptake was also observed in seminiferous tubules and epididymis of the intratesticular rete and the cauda epididymis groups, respectively. In conclusion, the combined approach of TMGT and lipofection can lead to ovine sperm transfection. This approach has potential to be combined with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas technology and used for on-farm gene editing of sheep species. پرونده مقاله

The fecundity in sheep has been much interested for producers due to its economical importance. Growth differentiation factor 9 (GDF9) is an autosomal gene with over dominance effect for twosingle nucleotide polymorphisms(SNPs), which resulted in sterility in homozygote mutant ewes and additive effect for three SNPs, without showing sterility event. In the present study, DNA was extracted from whole blood of 19 Iranian Afshari and 8 Shal sheep breeds. The extracted DNA was multiplied several folds for G1, G4 and G8 sites in GDF9 gene through polymerase chain reaction(PCR) technique and was sent for sequencing. Results showed that G1-GDF9 mutation (arginine to histidine shift) was detected in Afshari breed, while G4-GDF9 mutation (glutamic acid to lysine shift) was detected in both Afshari and Shal breeds. The observed SNPs were recorded with very low frequency (<8%) in fertile ewes with twining birth, while infertile ewe did not carry the mutant allele. This might indicate an association between these mutations and twin-births in Iranian breeds. Furthermore, these mutations could not be the reason for ewe infertility. In consistent with the rare frequency of infertility in Iranian sheep, the investigated population lacked the G8 or Thoka mutations, which are the most effective mutations in GDF9 gene.This may be due to lack or rare frequency of this SNP in Iranian sheep flocks. In conclusion, G1- and G4-GDF9 SNPs are segregating with very low frequency in Iranian sheep flocks, so that no homozygous ewe for these SNPs was detected in the current study. The same phenomenon could be the case for other important mutations and the sample size should be very large to rule out presence of such mutations in Iranian sheep breeds. پرونده مقاله