CRISPR/CAS9 MEDIATED EDITING OF COX-2 GENE IN LUTEAL CELLS OF BUFFALO
| dc.contributor.advisor | D. N. DAS | |
| dc.contributor.author | ARTHIYA K | |
| dc.date.accessioned | 2023-11-21T12:39:07Z | |
| dc.date.available | 2023-11-21T12:39:07Z | |
| dc.date.issued | 2022 | |
| dc.description.abstract | The domestic water buffalo (Bubalus bubalis) plays an important role in the economy of India as well as other developing countries in Asia by providing milk, meat and draught power and it has great unexploited production potential. One of the major constraints in the exploitation of the production potential of buffalo in comparison to cattle has been its inherently poorer reproductive efficiency that is mainly due to fertilization failure and early embryonic mortality. In many cases early embryonic mortality occurs because of luteal insufficiency. Corpus luteum plays a central role in maintenance of pregnancy, therefore it is necessary to prevent regression of CL for successful completion of pregnancy. One of the Prostaglandin (PG) i.e. PGF2α is the primary luteolysin in ruminants which causes regression of corpus luteum. The production of PGs is mainly governed by the rate limiting enzyme viz., cyclooxygenase-2 (COX-2) which is responsible for the conversion of arachidonic acid into PGH2, a common precursor of the various forms of PGs including PGF2α and PGE2. The present study was designed to examine the mRNA expression of edited COX-2 gene in the luteal cells of buffalo and it was undertaken with three objectives. Under objective 1: The CL tissues were assigned to consecutive stages viz., early, mid and late. Total RNA was isolated from these targeted tissue samples of early, mid and late stage. A total of six qRT-PCR experiments were conducted using corpus luteum samples. In this experiments, 5 samples of each stage were analysed keeping early stage as control. The qRT-PCR results revealed the mRNA expression of COX-2 in CL during the early luteal phase was higher followed by a continuous and significant downregulation afterwards. Under objective 2 and 3: Two single guide RNA against exon 1 were designed for COX-2 gene located at chromosome 5. The sgRNAs were successfully cloned into CRISPR/Cas9 plasmid vector separately and validated by PCR amplification using U6 primers. Analysis of sequence confirmed the cloning of sgRNAs into the vector. The resultant CRISPR/Cas9-sgRNA constructs were subsequently used for Lipofectamine mediated transfection into in-vitro cultured buffalo luteal cells. The cells transfected without CRISPR/Cas9-sgRNA construct was used as control. Puromycin was used for screening of the transfected cells. CRISPR/Cas9 tool was used for editing COX-2 gene using each sgRNA separately. Total RNA was isolated from the puromycin selected cells to investigate the effect of CRISPR/Cas9 mediated editing of COX-2 gene on its mRNA expression. The qRT-PCR results revealed the significant decline in COX-2 mRNA expression in the edited samples in comparison to the control. Genomic DNA extracted from the edited cell samples transfected with CRISPR/Cas9-sgRNA 1 construct along with control samples were subjected to PCR amplification using COX-2 gene specific primers. PCR amplified product was cloned using Topo TA cloning vector and the E. coli (DH5α strain) transformants were screened by ampicillin resistance. A total of 2 colonies were observed and plasmid DNA was isolated from these colonies. PCR amplification of plasmid DNA was performed using M13 primers. Out of the two colonies observed one was found to be positive clone. On the basis of above findings, it is concluded that the targeted editing of COX-2 gene significantly declined the mRNA expression of COX-2 gene in in-vitro cultured buffalo luteal cells. | |
| dc.identifier.uri | https://krishikosh.egranth.ac.in/handle/1/5810201635 | |
| dc.language.iso | English | |
| dc.pages | 96 p. | |
| dc.publisher | ICAR-SRS-NDRI, KARNAL | |
| dc.sub | Animal Genetics and Breeding | |
| dc.theme | ANIMAL GENETICS & BREEDING | |
| dc.these.type | M.V.Sc. | |
| dc.title | CRISPR/CAS9 MEDIATED EDITING OF COX-2 GENE IN LUTEAL CELLS OF BUFFALO | |
| dc.type | Thesis |