Date of Award

12-2009

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Animal and Veterinary Sciences

Advisor

Pratt, Scott L

Committee Member

Gibbons , John R

Committee Member

Ellis , Steve E

Abstract

In vitro-produced embryos exhibit aberrations in development, but the reasons for these developmental problems are unknown. Recently, a class of small non-coding RNA called microRNA (miRNA) has been described and reported to have roles in normal mammalian embryonic development. These miRNAs are encoded in the genome, transcribed by RNA pol II and processed into fragments approximately 22 nt in length by ribonuclease enzymes, the final one being a protein called Dicer. miRNA work through the RNA-induced silencing complex (RISC), of which the argonaute gene family are key proteins. Argonaute-2 (Ago2) has been identified as the only member possessing endonuclease activity, which is responsible for the breakdown of the miRNA target message. The cDNA sequences for Dicer and Ago2 have yet to be identified in pigs. Our objective is to identify the cDNA sequence for porcine Dicer (pDicer) and Ago2 (pAgo2) and verify their expression in reproductive tissue. A PCR cloning strategy was implemented, using over-lapping primers generated to highly conserved nucleotide sequences of Dicer and Ago2 known from other species. tcRNA was isolated from porcine ovaries and subjected to endpoint RT-PCR. To generate PCR primers, the cDNA sequences for bovine, human, and mouse Dicer and Ago2 were aligned; primers were generated from highly conserved regions using the Vector NTI program. Eight primer sets were designed for overlapping fragments of the pDicer sequence, and five primer sets were designed for pAgo2. PCR reactions were visualized using slab gel electrophoresis, EtBr staining, and UV-light exposure; after which, they were subcloned into the pDrive Cloning Vector and subjected to dideoxy-sequencing at the Clemson University Genomics Institute. Sequences were submitted to BLAST to verify each fragment as pDicer or pAgo2. Respective sequence fragments were assembled to generate the complete coding cDNA sequence for pDicer and pAgo2. Sequencing revealed two possible splice variants for pAgo2. Two additional primer sets were designed to confirm these splice variant sequences. The obtained pDicer sequence is 5,995 nt, contains the entire coding region, and exhibits a sequence identity of 91% to bovine, 90% to human, and 86% to mouse Dicer sequences. The obtained pAgo2 nucleotide sequence is 2,703 nt with a sequence identity of 94.2% to bovine, 92.2% to human, and 89.4% to mouse Dicer sequences. The sequencing data also indicate two possible splice variants of pAgo2, which could indicate the presence of as yet unidentified Ago2 variants in other species. Altogether, the data indicate that Dicer and Ago2 are present in porcine ovary and that the sequences are highly similar to those reported for other species. In addition, endpoint RT-PCR indicates that both Dicer and Ago2 are present in porcine embryos during early embryonic development, suggesting a role for miRNA in early embryonic development in pigs.

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