Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes.
The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein (EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5%±2.2% v.s. 22.9%±2.9%). Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer (NT). Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.
Department of Animal Sciences, Chungbuk National University, Republic of Korea.
Yong-Nan
YN
Xu
Sang-Jun
SJ
Uhm
Bon-Chul
BC
Koo
Mo-Sun
MS
Kwon
Ji-Yeol
JY
Roh
Jung-Seok
JS
Yang
Hyun-Yong
HY
Choi
Young-Tae
YT
Heo
Xiang-Shun
XS
Cui
Joon-Ho
JH
Yoon
Dae-Hwan
DH
Ko
Teoan
T
Kim
Nam-Hyung
NH
Kim
D000818:Animals; D002417:Cattle; D002460:Cell Line; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D016089:Immunodeficiency Virus, Feline / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism*; D014161:Transduction, Genetic; D015053:Zygote / Q000166:cytology / Q000378:metabolism
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins