The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer.
In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.
Nakauchi Stem Cell and Organ Regeneration Project, Japan Science and Technology Agency (JST), ERATO, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Masahito
M
Watanabe
Mayuko
M
Kurome
Hitomi
H
Matsunari
Kazuaki
K
Nakano
Kazuhiro
K
Umeyema
Akira
A
Shiota
Hiromitsu
H
Nakauchi
Hiroshi
H
Nagashima
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D046149:Embryo Culture Techniques; D018014:Gene Transfer Techniques; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D000075462:Serum Albumin, Human; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods*; D013552:Swine / Q000235:genetics*; D019076:Transgenes
C422455:ALB protein, human; D024042:Collagen Type I; D012709:Serum Albumin; D000075462:Serum Albumin, Human