title: Production of transgenic pigs mediated by pseudotyped lentivirus and sperm.
abstract: Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency.
authors.affiliation: Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Qianyun
authors.initials: Q
authors.lastname: Xi
authors.forename: Jinghua
authors.initials: J
authors.lastname: Ding
authors.forename: Weiguang
authors.initials: W
authors.lastname: Cai
authors.forename: Fanmin
authors.initials: F
authors.lastname: Meng
authors.forename: Junyun
authors.initials: J
authors.lastname: Zhou
authors.forename: Hongyi
authors.initials: H
authors.lastname: Li
authors.forename: Qingyan
authors.initials: Q
authors.lastname: Jiang
authors.forename: Gang
authors.initials: G
authors.lastname: Shu
authors.forename: Songbo
authors.initials: S
authors.lastname: Wang
authors.forename: Xiaotong
authors.initials: X
authors.lastname: Zhu
authors.forename: Ping
authors.initials: P
authors.lastname: Gao
authors.forename: Zhenfang
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015139:Blotting, Southern; D004431:Ear, External / Q000378:metabolism; D005260:Female; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007315:Insemination, Artificial; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D009928:Organ Specificity; D013094:Spermatozoa / Q000821:virology*; D013552:Swine / Q000235:genetics*; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins