Abstract
This study examined the efficiency of an osmotic differential to promote the influx of extracellular medium and hence the uptake of foreign DNA into common 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 solution (590 mOsm/kg) and subsequently rehydrated with a hyposmotic solution (130 mOsm/kg) containing pRSV-CAT DNA to regain the original osmotic pressure of the seminal fluid (about 300 mOsm/kg). Electroporation (pRSV-CAT DNA concentration of 30 ng/μl, field strength of 3.5 kV/cm, pulse length of 500 μs) was applied during the process of rehydration. The success rates of gene transfer assessed in 30-day-old fish were 66% for electroporation during the rehydration process of dehydrated sperm, 8% for electroporation in isosmotic conditions, and 20% for electroporation in hyperosmotic conditions. These results suggest that electroporation during rehydration of dehydrated sperm can greatly enhance the uptake of foreign DNA leading to increased gene transfer frequencies.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call