Transgenic-Cloned Pigs Systemically Expressing Red Fluorescent Protein, Kusabira-Orange

Abstract

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.

Cloning of Homozygous a1,3-Galactosyltransferase Gene Knock-Out Pigs by Somatic Cell Nuclear Transfer

“…The overall production efficiency of the cloned piglets was 3.1% from the recipients that farrowed or received a Caesarian section (17/547). Cloning efficiency of GalT-KO pigs in these experiments was comparable to that of non-KO pigs that we had reported previously (Kurome et al, 2006;Matsunari et al, 2008b).…”

“…Gal-T KO* Transgenes** sex DK3-1 homozygous hDAF and GnT-III male DK3-2 homozygous hDAF and GnT-III female DK3-9 homozygous NIL female *α1,3-galactosyltransferase gene knock out ** hDAF: decay-accelerating factor ; GnT-III: N-acetylglucosaminyltransferase III (Faast et al, 2006;Kurome et al, 2008b;Matsunari et al, 2008b). For the cloning of GalT-KO pigs in this study, we used preadipocytes (Tomii et al, 2005;Tomii et al, 2009), which have consistently produced good results in somatic cell cloning in our laboratory.…”

Xenotransplanted Embryonic Kidney Provides a Niche for Endogenous Mesenchymal Stem Cell Differentiation into Erythropoietin-Producing Tissue

“…Kusabira-Orange Tg swine were established as described elsewhere [23]. Their MSCs were processed using the same protocol for MSC derivation from human adipose tissue [24] and cryopreserved in a-modified Eagle's medium (a-MEM; Gibco-BRL, Tokyo) supplemented with 10% FBS, 1% antibiotic-antimycotic (Gibco-BRL), and 10% dimethyl sulfoxide (DMSO; Merck, Tokyo).…”

Urine excretion strategy for stem cell-generated embryonic kidneys

“…SCNT was conducted as described previously (8,18), using in vitromatured oocytes as the recipient cytoplasts. Primary culture cells of porcine fetal fibroblasts (female) were prepared as nuclear donors after cell-cycle synchronization, which was accomplished using serum starvation (FBS, 0.5% vol/vol) for 48 h. A single donor cell was inserted into the perivitelline space of an enucleated oocyte.…”