Detection of osteopontin gene transcripts in bull spermatozoa
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Date
2020-02
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Abstract
The present experiment was aimed to detect osteopontin gene transcript from bull spermatozoa. Fresh semen samples from 12 Jersey cross breed bulls were collected using artificial vagina. The volume and concentration of the individual samples were recorded. The spermatozoa were separated from bull semen samples by swim up protocol using sperm TALP. The initial concentration of semen sample was checked immediately before proceeding for RNA isolation and normalization of initial concentration was done. So, initial amount of every sample was made equal. Total RNA from the bull spermatozoa were extracted by the RNeasy® Mini Kit, Qiagen as per manufacturer’s protocol. The purity and concentration of the samples were measured at A280/260 by using Nano Drop TM 1000 spectrophotometer. The first strand cDNA was synthesized from 1μg total RNA using M-Mu LV Reverse Transcriptase Revert Aid TM H minus first strand cDNA synthesis kit (Thermo Scientific) as per manufacturer’s protocol. PCR product of about 267 bp fragment length of OPN gene was confirmed by conventional PCR. The bulk PCR product of about 100μl was purified by Gen Elute™ PCR Clean-Up Kit Sigma-Aldrich as per manufacturer’s protocol. The concentration and purity of PCR product were 64ng/μl and 1.6 respectively. The cloning and expression of OPN gene was carried out by using TA cloning kit. The ligated product was then transformed into E. coli DH5α cells. The plate was incubated for overnight at 37 ˚C for selection of the colonies and stock cultures were maintained in LB media at -80˚ C. The clones which showed the amplification of 400 bp DNA fragment were considered as positive clone carrying desired insert. Plasmid isolation was done as per the protocol followed in Hi Yield TM Plasmid Mini Kit (RBC Cat. NO. YPD100) and the eluted DNA were stored at -20 ˚C. The concentration and purity of PCR purified product were 67ng/μl and 1.6 respectively. The eluted plasmid DNA was sequenced commercially (Amnion, Bangalore) using M13 primers. The cloned partial sequence of OPN gene isolated from bovine spermatozoa was submitted at DDBJ (Accession No. AB983656). On blast analysis using NCBI nucleotide blast (Online tool), 99 per cent identity was found with reported consensus sequences except single nucleotide variation in DNA sequence at 234th nucleotide. There was substitution of adenine instead of cytosine.
Description
TNV_IJCS_2020_8(2)649-655
Keywords
Veterinary Science