Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.
Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.
Kawada
T
T
The Second Department of Internal Medicine, Health Service Center, Tokyo, Japan.
Shin
W S
WS
Nakatsuru
Y
Y
Koizumi
T
T
Sakamoto
A
A
Nakajima
T
T
Okai-Matsuo
Y
Y
Nakazawa
M
M
Sato
H
H
Ishikawa
T
T
Toyo-Oka
T
T
D000818:Animals; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006321:Heart / Q000821:virology*; D006651:Histocytochemistry; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008297:Male; D008856:Microscopy, Fluorescence; D009206:Myocardium / Q000378:metabolism; D011510:Proteolipids / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D018108:Respirovirus / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D008164:Luminescent Proteins; D011510:Proteolipids; C020485:proteoliposomes; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase