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 efshα and efshβ 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 efshβ 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 eFSHα and eFSHβ 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.
Methods: A comparative cross-sectional study involving 86 Malay premature babies (ROP = 41 and non-ROP = 45) was performed from September 2012 to December 2014. Mutation analyses in (FEVR)-causing genes (NDP, FZD4, LRP5, and TSPAN12) were performed using DNA from premature babies using polymerase chain reaction (PCR) and direct sequencing. Sequencing results were confirmed with PCR-Restriction Fragment Length Polymorphism (RFLP).
Results: We found variants of FZD4, LRP5, and TSPAN12 in this study. One patient from each group showed a non-synonymous alteration in FZD4, c.502C>T (p.P168S). A synonymous variant of LRP5 [c.3357G>A (p.V1119V)] was found in 30 ROP and 28 non-ROP patients. Two variants of TSPAN12, c.765G>T (p.P255P) and c.*39C>T (3'UTR), were also recorded (29 and 21 in ROP, 33 and 26 in non-ROP, respectively). Gestational age and birth weight were found to be significantly associated with ROP (P value < 0.001 and 0.001, respectively).
Conclusion: Analysis of data obtained from the ROP Malay population will enhance our understanding of these FEVR-causing gene variants. The c.3357G>A (p.V1119V) variant of LRP5, and c.765G>T (p.P255P) and c.*39C>T variants of TSPAN12 could be common polymorphisms in the Malay ethnic group; however, this requires further elucidation. Future studies using larger groups and higher numbers of advanced cases are necessary to evaluate the relationship between FEVR-causing gene variants and the risk of ROP susceptibility in Malaysian infants.