Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange.
Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.
Matsunari
Hitomi
H
Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan.
Onodera
Masafumi
M
Tada
Norihiro
N
Mochizuki
Hideki
H
Karasawa
Satoshi
S
Haruyama
Erika
E
Nakayama
Naoki
N
Saito
Hitoshi
H
Ueno
Satoshi
S
Kurome
Mayuko
M
Miyawaki
Atsushi
A
Nagashima
Hiroshi
H
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D019976:Cloning, Organism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D049408:Luminescent Agents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D011247:Pregnancy; D013552:Swine; D014018:Tissue Distribution; D000097573:Red Fluorescent Protein
D049408:Luminescent Agents; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein