Cloning of DNA sequences encoding RSVP14, RSVP20 and RSVP22 proteins from ovine seminal plasma | Clonaje de las secuencias que codifican para las proteínas RSVP14, RSVP20 y RSVP22 del plasma seminal ovino
2011
Serrano, E., Universidad de Zaragoza (España). Facultad de Veterinaria | Beamonte, R., Universidad de Zaragoza (España). Facultad de Veterinaria | Guillén, N., Universidad de Zaragoza (España). Facultad de Veterinaria | Calleja, L., Universidad de Zaragoza (España). Facultad de Veterinaria | Pérez Pé, R., Universidad de Zaragoza (España). Facultad de Veterinaria | Muiño Blanco, T., Universidad de Zaragoza (España). Facultad de Veterinaria | Cebrián Pérez, J.A., Universidad de Zaragoza (España). Facultad de Veterinaria
The adsorption of seminal plasma proteins (SPP) to the ram sperm surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. Previously, we have reported that three ram SPP, RSVP14, RSVP20 and RSVP22 are responsible for this protective effect. They can both prevent and repair the coldshock damage on the sperm membrane surface. Obtaining these proteins in vitro would allow us to formulate better cryopreservation diluents. In this study, we present the polymerase chain reaction (PCR) profile necessary to amplify the DNA sequences that codify for these proteins and its inclusion into plasmids. RNA extracted from the seminal vesicles of Rasa Aragonesa rams was retrotranscribed to obtain cDNA, which was used in order to carry out the PCR reactions. Primers were designed for each sequence. Direct sequencing confirmed that cycling conditions of 94 deg C 1 minute; [94 deg C-45 seconds; 54 deg C-45 seconds; 72 deg C-1 minute] x 30; 72 deg C-5 minutes for RSVP20 and RSVP22 are the most appropriate conditions to obtain the desired sequences and that RSVP14 can only be amplified using a touchdown PCR protocol with cycling conditions of 94 deg C-2 minutes; [94 deg C-15 seconds; from 70 deg C to 40 deg C (decreasing the temperature one grade each cycle)-30 seconds; 70 deg C-30 seconds] x 30; [94 deg C-15 seconds, 50 deg C-30 seconds, 70 deg C-30 seconds] x 10, 72 deg C-5 minutes.
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Este registro bibliográfico ha sido proporcionado por Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria