Development of a positive method for male stem cell-mediated gene transfer in mouse and pig

• Published in: Molecular reproduction and development Vol. 46; no. 4; pp. 515 - 526
• Main Authors: Kim, J.H. (Kon-Kuk University, Seoul, Korea.), Jung-Ha, H.S, Lee, H.T, Chung, K.S
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-04-1997; Wiley-Liss
• Subjects: ANIMAL TRANSGENIQUE; ANIMALES TRANSGENICOS; Animals; Animals, Genetically Modified; Biological and medical sciences; Biotechnology; CERDO; DESARROLLO EMBRIONARIO; DEVELOPPEMENT EMBRYONNAIRE; DNA - genetics; EMBRYONIC DEVELOPMENT; EMBRYONIC STEM CELLS; ESPERMATOZOO; Female; Fertilization in Vitro; Fundamental and applied biological sciences. Psychology; GENE TRANSFER; Gene Transfer Techniques; Genetic engineering; Genetic Markers; Genetic technics; LacZ; liposome; Male; male stem cells; Methods. Procedures. Technologies; MICE; Mice, Inbred ICR; Microinjections; mouse; pig; PORCIN; RATON; Seminiferous Tubules - cytology; SOURIS; sperm vector; SPERMATOZOA; Spermatozoa - cytology; Spermatozoa - transplantation; SPERMATOZOIDE; Stem Cell Transplantation; Stem Cells - cytology; SWINE; testis; TRANSFERENCIA DE GENES; TRANSFERT DE GENE; transgenic; TRANSGENIC ANIMALS; Transgenic animals and transgenic plants; Genetic transformation; Stem cell; Rodentia; Transgenic animal; Liposome; Male; Germinal cell; Testicle; Male genital system; Pig; Vertebrata; Mammalia; Transfection; Mouse; Genetic engineering; Artiodactyla; Technique; Ungulata
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/(SICI)1098-2795(199704)46:4<515::AID-MRD10>3.0.CO;2-V

Classical approaches for producing transgenic livestock require labor‐intensive, time‐consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994: Proc Natl Acad Sci 91:11298‐11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303‐11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short‐term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate‐buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0–14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7–13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome‐injected testes were all negative for X‐gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3–25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock. Mol. Reprod. Dev. 46:515–526, 1997. © 1997 Wiley‐Liss, Inc.