Dynamics of interaction between ram sperm with plasmid carrying human lysozyme gene in SMGT

Subject Areas : Journal of Comparative Pathobiology

Hoseini Pajooh, K.,Tajik,P.*, Karimipour,M. . ¹

1 -

Received: 2014-04-25 Accepted : 2014-09-07 Published : 2015-01-21

Keywords: Sperm, Sheep, Gene Transfer, Human Lysozyme,

Abstract :

Many studies have shown spermatozoa can be used as vectors for transfer of exogenous DNA tooocyte to produce transgenic animals. However its efficiency is still questionable. The results ofDNA concentration and incubation time on DNA uptake and sperm functionality has beendifferent or sometimes opposite. In this study the effect of DNA concentration and incubationtime of sheep spermatozoa with foreign DNA on DNA uptake, its dynamic and spermfunctionality and their difference between ejaculated and epididimal sperm was investigated.For this the ejaculated(N=4) and epididimal(N=6)spermatozoa was incubated with differentconcentration (5-500ng/106 sperm/25μl) of rhodamine labaled pEGFP-IRES-hLys in varioustime (15-90min) and DNA uptake, dynamic of uptake and sperm functionality was assayed.Results showed to an average of 65% of incubated spermatozoa uptake the foreign DNA,depend on DNA concentration and incubation time. Although the amount of transfectedspermatozoa could increase significantly with prolonged incubation time up to 60 min but theamount of DNA uptake was higher in the first 15min of incubation. Increase of foreign DNAconcentration and incubation time increased the transfected spermatozoa and uptake intensity. Itseems higher DNA concentration, more rapid DNA uptake and more rapid gain of transfectionpeak. Slight decrease of sperm total and progressive motility was seen in treatment group incomparison with Control groups (except in 5min incubation). No spermatozoa with postacrosomal absorption (true uptake) were motile. So incubation for more than 30 and 60 min inmoderate (20-200ng) and low (5ng) plasmid concentration respectively is not useful.