5013
WALEID MOHAMED EL-SAYED SHAKWEER
PHYSIOLOGICAL AND GENETICAL STUDIES ON REPRODUCTION OF SHEEP
Growth hormone (GH), Orthologous genes, Egyptian x Holstein crossbred cows, Ossimi sheep breed, sperm uptake, pmKate2-N expression vector
The aim of this study is to produce transgenic ovine spermatozoa carrying either bovine or ovine growth hormone (GH) cDNA sequence of Egyptian x Holstein cows or Ossimi sheep. This study has been conducted as an introduction to produce either bovine or ovine GH-transgenic sheep using sperm mediated gene transfer (SMGT) technique to improve their production. The mRNA of pituitary gland has been isolated and reversed to cDNA which cloned into pTZ57R/T cloning vector and then into pmKate2-N expression vector. The GH gene of Egyptian x Holstein crossbred (EH_GH) and Ossimi sheep breed (Os_GH) has been sequenced and characterized in this study. A total of 160 μl ovine sperm cell suspensions (40 x 106 final concentrations) have been incubated with 200 ng of pmKate2-N expression vector. Total of nine experiments have been conducted; four groups for Os_GH-pmkate2-N vector, four groups for EH_GH-pmkate2-N vector and control as following; 1). Control, ovine spermatozoa have been incubated in the absence of pmKate2 vector at 37◦C for 1h., 2). 37◦C for 1h group, ovine spermatozoa have been incubated with pmkate2-N vector at 37◦C for 1h. 3). Heat shock group, ovine spermatozoa have been incubated with pmkate2-N vector at 4◦C for 20 min and then 42◦C for 2 min. 4). Heat shock + DMSO group, ovine spermatozoa have been supplemented with 3% DMSO and incubated with pmkate2-N vector at 4◦C for 20 min and then 42◦C for 2 min. 5). DMSO group, ovine spermatozoa have been supplemented with 3% DMSO and incubated with pmkate2-N vector at 37◦C for 1h. The complete coding sequences (CDS) of EH_GH and Os_GH were detected, annotated comprehensively and registered in Gene Bank under accession number AC: KP221576 and Ac: KP221575, respectively. The sequenced GH genes varied slightly at gene length; EH_GH was 740 bp instead of 690 bp in Os_GH, but with similar gene structure. High homology (98%) in GH protein length and structure was observed; the predicted open reading frames (ORF) length was 217 aa and 216 aa for EG_GH and Os_GH, respectively. A gene synteny was developed for the homologous bovine and ovine GH1 genes Ensemble annotated genome database. The results of geneannotation showed strong conserved structure and function that indicated that GH gene of Egyptian x Holstein crossbred cows and Ossimi sheep breed are orthologous genes. The fluorescent fusion protein in both EH_GH or Os_GH-pmKate2-N vector have been expressed in transformed DH10B cells with expression of 24558 Da for EH_GH protein and 24460 Da for Os_GH protein. The EH_GH or Os_GH-pmKate2-N vector was uptake efficiently by spermatozoa in all treated groups. The novel genetic information about the bovine and ovine GH in Egyptian breeds and crossbred and ability of sperm uptake that can support possibility of producing the bovine or ovine GH-transgenic sheep to improve the growth and milk production.
2015
Ph.d
Cairo
Agriculture