Sperm Mediated Transgenesis Rushes ‘Back to the Future’
Authors: Jose B. Cibelli
Journal: Molecular Therapy (2002)

While a constant effort to increase human organ donation in the US and throughout the western world is made, many patients die every year waiting for a transplant. Pig organs are currently thought to be a valid alternative to fulfill this increasing demand. Still, a series of immunological barriers such as hyperacute reaction, a phenomenon produced as a consequence of complement activation, must be overcome. Lavitrano and colleagues have recently published a report describing the generation of transgenic pigs carrying the human Decay Accelerating Factor (hDAF) [1Lavitrano M. Bacci M.L. Forni M. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation.Proc. Natl. Acad. Sci. USA. 2002; 99: 14230-14235Crossref PubMed Scopus (152) Google Scholar]. hDAF is a membrane-bound protein that prevents the assembly of C3 convertases—thereby inhibiting complement activation [2Sun X. Funk C.D. Deng C. Sahu A. Lambris J.D. Song W.C. Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.Proc. Natl. Acad. Sci. USA. 1999; 96: 628-633Crossref PubMed Scopus (137) Google Scholar]. In the new study, published in the Proceeding of the National Academy of Sciences, transgenic pigs were able to transcribe and translate hDAF in a physiological manner. When the hearts of these transgenic animals were perfused ex vivo with human blood, they ‘functioned longer and better and with less damage’ than non-transgenic control hearts. These remarkable pigs expressing functional hDAF will certainly play a role in the field of transplantation medicine. However, the real breakthrough in Lavitrano's work resides in the elegant and simple approach used to generate the transgenic pigs. Many different strategies have been developed to create an animal carrying foreign DNA stably integrated into its genome and capable of transmitting that DNA to their offspring. The most widely used procedures include pronuclear microinjection, virus-mediated transduction, nuclear transplantation, and at the experimental level, germ cell transplantation [3Wall R.J. New gene transfer methods.Theriogenology. 2002; 57: 189-201Abstract Full Text PDF PubMed Scopus (83) Google Scholar]. In pigs, nuclear transfer using transgenic somatic cells or pronuclear microinjection are the methods of choice. Unfortunately, both methods are rather inefficient. While transgenic somatic cell (cloning) allows us to make sophisticated genetic manipulations such as gene knockouts, its low efficiency and requirement for extensive embryonic manipulation are genuine drawbacks. Pronuclear microinjection is widely used in mice to over express specific proteins, yet, in swine zygotes, additional modifications to the technique are required that in turn yield low transfection efficiencies of somewhere between 0.5 to 5%. It was 1989, and eighteen years after the original description of sperm-mediated DNA transfer by Brackett et al., when Lavitrano and colleagues first published an efficient way to create transgenic mice by introducing foreign DNA into an egg via the sperm. Attempts to replicate this work were made in different laboratories and in different species with mixed results. Now, 13 years later, Lavitrano and her group seem to have settled the case for good: sperm mediated transgenesis, when performed properly, can work wonders [1Lavitrano M. Bacci M.L. Forni M. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation.Proc. Natl. Acad. Sci. USA. 2002; 99: 14230-14235Crossref PubMed Scopus (152) Google Scholar]. Lavitrano and colleagues’ simple technique to make transgenic pigs does not require any embryonic manipulation. They carefully removed all the non cellular components from freshly ejaculated semen, and incubated the sperm with linearized DNA plasmid for 2 hours at 17°C followed by 20 minutes at room temperature and a final one minute incubation at 37°C. This semen was then used for artificial insemination using standard procedures. The results were remarkable. 53 out of 93 animals born were transgenic (57%) and 34 of them transcribed the transgene properly. More than 80 of the founders in which mRNA was detected expressed the protein at a level similar to that found in normal human heart. Overall, almost a third of the animals born expressed the protein. It is important to note that Lavitrano's protocol is not based upon the use of liposomes or electroporation to incorporate the DNA into the sperm—the mere incubation of ‘clean’ spermatozoa with naked plasmids seem to do the trick. Certain aspects of the mechanism by which the sperm is able not only to carry the gene inside the egg, but also to incorporate it stably into the genome of the new animal are the subject of current investigation. This same group has previously shown that the sperm must be thoroughly cleaned in order to remove DNA uptake-inhibitory proteins present in the ejaculate. This is perhaps a conserved mechanism by which the sperm protects itself from undesired DNA contaminants [4Spadafora C. Sperm cells and foreign DNA: a controversial relation.Bioessays. 1998; 20: 955-964Crossref PubMed Scopus (126) Google Scholar]. But how does the foreign DNA bind to the sperm, and how does it then become internalized? It has been shown using knockout mice that the major histocompatibility complex class II is required for DNA binding to sperm and the CD4 protein seems to be important for the internalization of the DNA into the nucleus [5Lavitrano M. Maione B. Forte E. The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatibility complex class II molecules.Exp. Cell Res. 1997; 233: 56-62Crossref PubMed Scopus (78) Google Scholar]. What remains to be elucidated however, is the final target in the nucleus for these foreign DNA molecules. It is also unclear whether plasmids are transported directly into the egg as non-genomic DNA, or if they are stably integrated into the genome of the sperm before fertilization takes place. It has been shown that some of the DNA is indeed incorporated into the sperm genome but the same authors did not rule out the possibility that plasmid DNA is transferred directly into the egg [4Spadafora C. Sperm cells and foreign DNA: a controversial relation.Bioessays. 1998; 20: 955-964Crossref PubMed Scopus (126) Google Scholar]. In the first scenario the sperm would be responsible for the generation of the transgenic animal; in the second, the DNA would have to recombine with the genomic DNA of the egg during the early rounds of embryonic cell division—something that in turn could generate mosaic animals. An interesting aspect to consider from this study is the fact that only 2 out of 20 boars were used as sperm donors. Although no clear rational was given as to why these two animals were chosen over the rest, studies in mice have shown a clear variation between animals in their capacity to take up DNA. This feature would perhaps help elucidate those aspects of the mechanism of sperm mediated transgenesis that remain unclear. It seems now reasonable to predict that this new/old technique may soon replace pronuclear microinjection and viral-mediated transgenesis; after all, there is no embryo manipulation required. Having a ‘user friendly’ technique for large animal transgenesis would certainly encourage more researchers to create large animal models for human disease without been intimidated by the prospects of using sophisticated laboratory techniques.