Models of Transgene Integration and Transmission

The discovery that spermatozoa of essentially all species can spontaneously take up exogenous DNA and deliver it to oocytes at fertilization suggested their use as vectors of foreign information for the generation of transgenic animals. Hence a variety of protocols of sperm-mediated gene transfer (SMGT) have been developed in numerous species. The outcomes in stability and transmission of the exogenous sequences are highly heterogeneous, casting doubt on whether the generated animals are truly transgenic and leaving an open question as to the final fate of the DNA molecules delivered by sperm cells. Two findings have contributed to clarify the underlying molecular mechanism of SMGT: i) the discovery that DNA-loaded demembranated spermatozoa used in ICSI assays increase the yield of genuine transgenic mice, and ii) the identification of a reverse transcriptase (RT) activity in mature spermatozoa. When membrane-disrupted sperm cells are incubated with DNA prior to microinjection in oocytes, the foreign sequences are integrated in the host genome and a high proportion of the offspring are genuinely transgenic. In intact spermatozoa, instead, the foreign DNA binds to the plasma membrane, hence is internalised and undergoes a two-step reaction, first of transcription in RNA and then of reverse-transcription in cDNA copies; these cDNAs behave as transcriptionally competent retrogenes, are propagated as non-integrated extrachromosomal structures and are transmitted to the progeny in a non-Mendelian fashion. We have called this phenomenon sperm-mediated “reverse” gene transfer (SMRGT). These results point out the central role of the plasma membrane in the final fate of exogenous sequences as either integrated transgenes or extrachromosomal retrogenes. The latter underscore a previously unrecognised transgenerational genetics, and a form of non-Mendelian inheritance, mediated by an RT-dependent mechanism.

Keywords: SMGT, Sperm, DNA binding, Sperm plasma membrane, Demembranated, Sperm cells, ICSI, Reverse Transcriptase, Episomal DNA, Retro-Genes, SMRGT, Non-Mendelian Genetics.