Partial complementary deoxyribonucleic acid cloning of equine relaxin messenger ribonucleic acid, and its localization within the equine placenta.

The research article discusses a process to identify the location of relaxin gene expression in equine placentae. They used specific primers and various other tools to analyse RNA from the placentae at different stages of pregnancy, confirming that the relaxin gene was expressed in the fetal trophoblast cells.

Research Process

The research employed a multi-step process to determine the location of relaxin gene expression in equine placentae:

• A set of degenerate oligonucleotide primers was created in accordance with the known amino acid sequence of the equine relaxin (eRXN).
• Total cellular RNA (tcRNA) from equine placentae at approximately 120 and 300 days of gestation was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using these primers.
• The amplified PCR output was detected through gel electrophoresis, which showed a single product of approximately 430 base pairs (bp) in each instance.

Confirmation of Cloned cDNA Fragment

• This PCR product was then ligated into a Bluescript plasmid and sequenced to verify the identity of the clone as an eRXN complementary DNA (cDNA) fragment.
• Nucleic acid sequence analysis further revealed this cloned fragment, measuring 428 bp, as an eRXN cDNA fragment that encoded parts of the A- and B-chain along with the connecting peptide (109 residues).

Use of Northern Analysis and In Situ Hybridization

• A northern analysis of tcRNA from placentae at 120 and 300 days of gestation was carried out using antisense digoxigenin-labeled cRNA generated from the cDNA clone; a single transcript of approximately 1 kilobase (kb) was found.
• In situ hybridization on placental tissue at 120 days and uteroplacental tissue at 300 days of gestation indicated that only the fetal trophoblastic cells expressed eRXN mRNA transcripts.
• The identity of these cells was confirmed with their positive staining with an antibody specific for equine trophoblast (cell surface) protein. The relaxin peptide was also detected in samples of the same placental tissues through immunohistochemical analysis.

Key Findings

• This study is the first to report the nucleic acid sequence of eRXN.
• Fetal trophoblast cells were identified as the site of eRXN mRNA expression and protein secretion in the equine placenta.