Cloning of a pig glucose transporter 4 cDNA fragment: use in developing a sensitive ribonuclease protection assay for quantifying low-abundance glucose transporter 4 mRNA in porcine adipose tissue.
A 246-bp fragment of porcine glucose transporter 4 (GLUT4) cDNA was cloned by polymerase chain reaction (PCR) from porcine adipose tissue RNA. Nucleotide sequences 1-138 and 139-246 of the GLUT4 cDNA share 78% sequence identity with exon 4a and 91% sequence identity with exon 4b of the human GLUT4 gene, respectively. The GLUT4 cDNA fragment was subcloned into pGEM-4Z vector to synthesize a highly specific riboprobe that hybridized only to human GLUT4 cDNA but not to human glucose transporter 1 (GLUT1) cDNA. Northern blot analysis of total RNA revealed the presence of a single transcript of 2.8 kb in porcine adipose tissue. Cloning a fragment of the GLUT4 cDNA enabled us to develop a ribonuclease protection assay for detecting porcine GLUT4 mRNA. The ribonuclease (RNase) protection assay is highly reproducible and retains a sensitivity level to as little as 2 pg of GLUT4 mRNA. The standard curve was linear between 2 and 128 pg of sense-strand GLUT4 RNA (r = .994). The ability to detect small quantities of GLUT4 mRNA is important when the abundance of GLUT4 mRNA is low and the quantity of tissue is limiting (e.g., when RNA is extracted from cultured adipose tissue). When porcine adipose tissue explants were cultured in the presence of insulin (10 ng/mL), GLUT4 mRNA abundance was increased. Development of a sensitive assay to quantify GLUT4 mRNA in porcine adipose tissue will enable us to conduct studies to increase our understanding of the molecular mechanisms by which porcine somatotropin (pST) regulates GLUT4 gene expression.
Chiu
P Y
PY
Department of Dairy and Animal Science, Pennsylvania State University, University Park 16802.
Chaudhuri
S
S
Harding
P A
PA
Kopchick
J J
JJ
Donkin
S
S
Etherton
T D
TD
D000273:Adipose Tissue / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D013552:Swine / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D004247:DNA