This is an unedited manuscript accepted for publication and provided as an Article in Press for early access at the author’s request. The article will undergo copyediting, typesetting, and galley proof review before final publication. Please be aware that errors may be identified during production that could affect the content. All legal disclaimers of the journal apply.
Journal Menu
By: Atul Khajuria and Ashish Kumar.
Faculty of Allied & Healthcare sciences, Rayat Bahra Professional University, Hoshiarpur
Spermmediated gene transfer (SMGT) exploits the natural role of spermatozoa as DNA vectors to deliver exogenous genetic material to oocytes, potentially enabling economical production of transgenic large animals without complex embryo micromanipulation (1). Early proofofconcept studies established that mammalian sperm can bind, internalize, and protect exogenous DNA, leading to integration, expression, and germline transmission in various species, including pigs (24). Subsequent mechanistic work has challenged the traditional view of sperm as metabolically inert, revealing regulated DNAbinding proteins on the sperm surface and retrotransposonencoded reverse transcriptase activity that can mediate reverse transcription and nonMendelian inheritance of exogenous sequences (3). In large animal models, SMGT has achieved high apparent rates of transgenesis in pigs, with reports of up to 80% of offspring harboring an integrated transgene and more than half expressing a functional protein (19). However, concerns remain regarding integration site heterogeneity, mosaicism, variable expression, and unintended genomic effects, which are now interrogated by highresolution molecular pathology tools, including nextgeneration sequencing and epigenomic profiling. This review revisits SMGT from the vantage point of pathology and molecular pathology, critically examining biological mechanisms, factors influencing uptake and integration, optimization strategies for delivery and germline transmission in large animals, and comparative performance against zygote microinjection, viral and CRISPRbased approaches. We also discuss potential applications in xenotransplantation, agriculture, and functional genomics, and outline a framework for histopathologic, genomic, and epigenomic safety assessment of SMGTderived lines.
Citation:
Refrences:
1. Lavitrano M, French D, Zani M, et al. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc Natl Acad Sci U S A. 2002;99(22):14230-5.
2. Chang K, Qian J, Jiang Y, et al. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer. Proc Natl Acad Sci U S A. 2002;99(23):14931-6.
3. Sciamanna I, Lorenz B, Randiglioni A, Spadafora C. Ongoing activity of reverse transcriptase in mature spermatozoa. J Cell Biol. 2000;148(6):1107-14.
4. Lavitrano M, Giovannangeli A, Ferranti P, et al. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell. 1989;59(5):850-71.
5. Brinster RL, Sandgren EP, Behringer RR, et al. No simple solution for making transgenic mice. Trends Genet. 1989;5(12):345-8.
6. Pramod RK, Kumar R, Mitra A, et al. Intratesticular injection followed by electroporation increases gene transfer efficiency in goat testis. Sci Rep. 2018;8(1):2775.
7. Lavitrano M, Stoppani C, Giancotti P, et al. Sperm-mediated gene transfer. Reprod Biomed Online. 2006;12(4):457-65.
8. Allen GC, Spiker S, Thompson WF. Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol Biol. 2000;43(5-6):361-76.
9. Scudamore E, McLean L, Mozzachiodi S, et al. Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps. Biol Reprod. 2005;73(6):1174-81.
10. Chang K, Qian J, Jiang Y, et al. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer. Proc Natl Acad Sci U S A. 2002;99(23):14931-6.
11. Sciamanna I, Lorenz B, Randiglioni A, Spadafora C. Reverse transcriptase activity in mature spermatozoa of mouse epididymis. J Cell Biol. 2000;148(6):1107-14.
12. Hughes SE, et al. Paternal effect of uncapped sperm telomeres in Drosophila. PLoS Genet. 2016;12(4):e1005973.
13. Perez LL, et al. Research article: sperm mediated gene transfer (SMGT). Recent Sci. 2004;83:1-12.
14. Arora S, et al. In vitro and in vivo optimization of liposomal nanoparticles for brain delivery of CRISPR/Cas9. Nanoscale. 2021;13(36):15458-73.
15. Lavitrano M, et al. The interaction between exogenous DNA and sperm cells. Mol Reprod Dev. 1992;32(1):139-45.
16. Arias ME, et al. Effect of transfection and co-incubation of bovine sperm with exogenous DNA on embryo development. Zygote. 2017;25(3):396-405.
17. Wakayama S, et al. Direct gene delivery to murine testis. J Reprod Dev. 2006;52(2):221-8.
18. Lavitrano M, et al. Efficient production by sperm-mediated gene transfer of hDAF transgenic pigs. PNAS. 2002;99(22):14230.
19. Spadafora C. Soma to germline inheritance of extrachromosomal genetic information via endogenous retroviruses. Bioessays. 2017;39(2).
20. Zani M, et al. The mechanism of binding of exogenous DNA to sperm cells. Hum Reprod. 1995;10(2):405-12.
21. Liu Y, et al. Screen and verification for transgene integration sites in pigs. Front Genet. 2018;9:197.
22. Yue Y, et al. CRISPR/Cas technology in pig-to-human xenotransplantation research. Front Immunol. 2021;12:666646.
23. Zamudio N, et al. Nuclease footprints in sperm project past and future chromatin regulatory events. Sci Rep. 2016;6:25864.
24. Wang Y, et al. Comparison of two methods of sperm- and testis-mediated gene transfer to produce transgenic animals. Anim Genet. 2024;55(2):134-46.
25. Wakayama S, et al. Direct gene delivery to murine testis. Reprod Med Biol.2006;5(2):221-8.
26. Wang Z, et al. Identification of signatures of selection by whole-genome sequencing in donkeys. Front Genet. 2020;11:566255.
27. Smolenski RT, et al. Reduction of hyperacute rejection by HMG-CoA reductase inhibitors in pig-to-human perfusion. Cardiovasc Res. 2007;73(1):143-52.
28. CAS. CRISPR could make xenotransplantation a success. CAS Insights. 2022.
29. Gardiner-Garden M, et al. Organization of sperm DNA by the nuclear matrix. Reprod Fertil Dev. 1998;10(1):1-11.
30. Rieth A, et al. Homologous recombination in bovine spermatozoa. Mol Reprod Dev. 2000;57(4):396-403.
31. Lavitrano M, et al. Efficient production by sperm-mediated gene transfer. EMBO J. 2003;22(4):974-82.
32. Killian GJ, et al. Seminal plasma proteins and metabolites: effects on sperm function. Biol Reprod. 2018;99(5):1025-42.
33. Spadafora C. Soma to germline inheritance via retroviruses. Bioessays. 2016;38(1):58-67
34. Celebi R, et al. Sperm-mediated gene transfer. Reprod Domest Anim. 2006;41(1):37-43.
35. Niemann H, Lucas-Hahn A. Somatic cell nuclear transfer in pigs. Theriogenology. 2012;78(8):1575-86.