This study was carried out the animal production department, genetic engineering lab, college of agriculture, （UoB）, Iraq. The aim of this study was to use the polymerase chain reaction restriction fragment length p...This study was carried out the animal production department, genetic engineering lab, college of agriculture, （UoB）, Iraq. The aim of this study was to use the polymerase chain reaction restriction fragment length polymorphism （PCR-RFLP） as a fast, efficient and low cost method to detect the genetic variants of kappa-casein gene （k-CN） in Iraqi buffalo using three different primers specific for bovine k-CN to amplify the gene segment, followed by digestion using restriction enzyme （Hind III） for genotyping. DNA from 50 Iraqi buffaloes was extracted by phenol chloroform method. PCR was carried out in a final reaction volume of 25 μL and the reaction mixture was subjected to standard PCR protocol. The results of this work show that among the examined 50 Iraqi Buffalo were homozygous for the K-CN and genotyped as BB for all three primers but gave different bands. Thus PCR-RFLP using Hind III revealed all the samples to be monomorphic for this locus. The restriction digestion analysis of 397 bp PCR product of k-CN indicates the presence of two fragments of 154 bp and 225 bp for BB-genotype. A 437 bp fragment of the bovine genomic K-CN gene was amplified. One Hind III restriction site is found in position 346 of the amplified fragment of allele k-CN B, yielded 91 bp and 346 bp. Amplified products from Iraqi buffalo （530）, after being digested with Hind III, yielded two separate DNA fragments of different sizes i.e., 160 bp and 370 bp. For the first time completed research such specifications in Iraq, for the first time using molecular biology in genetic identification. Our objectives of this study have been to aid in understanding domestication, Buffalo origin and their history and evolution, to identify genetically unique breeds, to provide an objective basis for conservation decisions and to aid the formulation of breeding plans.展开更多
Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were tran...Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and LacZ gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.展开更多
文摘This study was carried out the animal production department, genetic engineering lab, college of agriculture, （UoB）, Iraq. The aim of this study was to use the polymerase chain reaction restriction fragment length polymorphism （PCR-RFLP） as a fast, efficient and low cost method to detect the genetic variants of kappa-casein gene （k-CN） in Iraqi buffalo using three different primers specific for bovine k-CN to amplify the gene segment, followed by digestion using restriction enzyme （Hind III） for genotyping. DNA from 50 Iraqi buffaloes was extracted by phenol chloroform method. PCR was carried out in a final reaction volume of 25 μL and the reaction mixture was subjected to standard PCR protocol. The results of this work show that among the examined 50 Iraqi Buffalo were homozygous for the K-CN and genotyped as BB for all three primers but gave different bands. Thus PCR-RFLP using Hind III revealed all the samples to be monomorphic for this locus. The restriction digestion analysis of 397 bp PCR product of k-CN indicates the presence of two fragments of 154 bp and 225 bp for BB-genotype. A 437 bp fragment of the bovine genomic K-CN gene was amplified. One Hind III restriction site is found in position 346 of the amplified fragment of allele k-CN B, yielded 91 bp and 346 bp. Amplified products from Iraqi buffalo （530）, after being digested with Hind III, yielded two separate DNA fragments of different sizes i.e., 160 bp and 370 bp. For the first time completed research such specifications in Iraq, for the first time using molecular biology in genetic identification. Our objectives of this study have been to aid in understanding domestication, Buffalo origin and their history and evolution, to identify genetically unique breeds, to provide an objective basis for conservation decisions and to aid the formulation of breeding plans.
文摘Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and LacZ gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.
