ABSTRACT
Background: follicle stimulating hormone [FSH] plays an essential role in reproductive physiology and follicular development
Objective: a new variant of the equine fsh [efsh] gene was cloned, sequenced, and expressed in Pichia pastoris [P. pastoris] GS115 yeast expression system
Materials and Methods: the full-length cDNAs of the efsh[alpha] and efsh[beta] chains were amplified by reverse transcription polymerase chain reaction [RT-PCR] using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation [IP] methods
Results: the DNA sequence of the efsh[beta] chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3?UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSH[alpha] and eFSH[beta] subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit
Conclusions: eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares
ABSTRACT
Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk. In this experimental study, a linearized recombinant vector [pBC1] entailing human coagulation factor IX [5.7 kb] cDNA was introduced into goat fetal fibroblast cells [three sub passages] via electroporation in four separate experiments [while other variables were kept constant], beginning with 10 micro g DNA per pulse in the first experiment and increments of 10 micro g/pulse for the next three reactions. Thereafter, polymerase chain reaction [PCR]-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05. The results showed no significant difference among three first concentrations except for group 1 [10 micro g/pulse] and group 3 [30 micro g/pulse], but there was a significant difference between these groups and the fourth group [p<0.05], as this group [40 micro g/pulse] statistically showed the highest rate of transfection. As the fluorescence in situ hybridization [FISH] results indicated the transgene was integrated in a single position in PCR positive cells. Increasing amount of using the vector 40micro g/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency