Effect of an osmotic differential on the efficiency of gene transfer by electroporation of fish spermatozoa

This study examined the efficiency of an osmotic differential to promote th e influx of extracellular medium and hence the uptake of foreign DNA into c ommon carp sperm cells during electroporation. Carp spermatozoa diluted in a hyposmotic solution (< 290 mOsm/kg) become motile, but rapidly lose their fertility. Thus, spermatozoa were first dehydrated in a hyperosmotic solut ion (590 mOsm/kg) and subsequently rehydrated with a hyposmotic solution (1 30 mOsm/kg) containing pRSV-CAT DNA to regain the original osmotic pressure of the seminal fluid (about 300 mOsm/kg), Electroporation (pRSV-CAT DNA co ncentration of 30 ng/mu l, field strength of 3.5 kV/cm, pulse length of 500 mu s) was applied during the process of rehydration. The success rates of gene transfer assessed in 30-day-old fish were 66% for electroporation duri ng the rehydration process of dehydrated sperm, 8% for electroporation in i sosmotic conditions, and 20% for electroporation in hyperosmotic conditions . These results suggest that electroporation during rehydration of dehydrat ed sperm can greatly enhance the uptake of foreign DNA leading to increased gene transfer frequencies. (C) 1999 Elsevier Science B.V. All rights reser ved.