The Efficiency of Transgenesis by Restriction Enzyme Mediated Integration - Sperm Mediated Gene Transfer (REMI-SMGT)

Abstract

The present study tends to  test the validity of REMI-SMGT and to evaluate the efficiency of REMI-SMGT on generating other mammalian species rather than the sheep that made only by one group and to observe the possibility of doing so by using rabbits as a model for this approach and also to reduce the cost of REMI-SMGT by substituting liposomes and highly cost effective media with a high efficient, non-cost effective substitute.

Direct protective relationship of liposome with DNA and seminal fluid was identified compared with DMSO.  While different treatments (linearized DNA – restriction enzyme – liposome complex, DNA – restriction enzyme – DMSO complex, DNA –DMSO complex, DNA –liposome complex, and even naked DNA) were all found to be successful to internalize inside the head of the sperm according to PCR results, only three (one by restriction enzyme – liposome treatment and two by restriction enzyme – DMSO treatment) out of fourteen new born babies were found to be transgenic by PCR.

Despite the absolute ability of exogenous DNA to be internalized inside rabbit’s sperm head only few percent of transgenic babies were obtained. This may not reflect the weakness of restriction enzyme mediated transgenesis technique itself but it reflects the inability of recombinant sperm to fertilize superovulated oocyte compared with their normal counterparts. Comparable results were found between liposome and DMSO treatment which may reflect direct relationship of DMSO with the cell membrane instead of with the exogenous DNA itself as what is found with liposome.

Key words : REMI-SMGT , PCR , DNA – restriction enzyme , DMSO