Hello everyone,
I am doing an experiment in which, the plan of work is to electroporate a clone (plasmid form) into chicken spermatozoa, which will be used further for artificial insemination. Could anyone help me with the protocol to be followed like electroporation conditions and buffers to be used?
Thanking you in anticipation
Hi Rajendra
While I do not have any experience of working with chick sperms, I would suggest to optimize electroporation conditions first. For example, I use Neon nucleofection apparatus. I first did try various voltages /duration of voltage/ number of pulses to find a voltage with highest viability after electroporation and highest transfection efficiency (using GFP for example, you can qualitatively measure efficiency of electroporation). It is a trade off. You get higher transfection efficiency at high voltages but low viability and vice versa. You should find what is acceptable for your experiment.
AFTER that you can optimize DNA concentration which is optimum and then you can do real experiments with new cell line. goodluck!
pDNA will not integrate into sperm nucleus. You can use mangetofaction, nanofaction, electroporation or even lipofection to introduce the pDNA into sperm cells but it will not integrate.
Nevertheless, if your idea is to do SMGT, you can simply mix with purified sperm and do AI. But, it seems there is a technical know-how in getting the success. While it has been very successful in Lavitrano's lab, most other labs have failed. A number of papers have also shown detailed mechanism of DNA update by sperm and methods of its improvement. However, despite being attractive, most labs have failed.
Many labs have also used the pDNA mixed with sperm to do ICSI to produce Tg animals (See Anthony Perrys works) and it worked in may labs.
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