Expression of fibronectin isoforms in rat cornea after an epithelial-scrape wound.
The polymerase chain reaction (PCR) technique was used to analyze the presence or absence of the EIIIB and V region fibronectin (FN) mRNA isoforms, generated by alternative splicing of the FN primary mRNA transcript during epithelial wound healing, in a rat cornea wound model. A central 4mm area of the cornea was denuded of epithelium. At 5, 15, 30, 45 minutes, 1, 2, 4, 8, 24, 48 hours, and 4 days post-wounding, the rats were euthanized and a 6mm diameter full thickness corneal biopsy was performed. cDNA was synthesized from extracted RNA, and specific FN sequences were amplified by PCR as directed by different sets of 5' and 3' primers specific for the alternatively spliced EIIIB and V region domains. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used to normalize the data. PCR products were analyzed by agarose gel electrophoresis and the identity of the bands were confirmed by direct nucleotide sequencing. The alternatively-spliced EIIIB and all three alternatively spliced isoforms of V domain FN mRNA were observed post-wounding. Similarly, in normal tissue, EIIIB and all three V region alternatively spliced FN isoforms were expressed. These findings suggest that in situ synthesis of cellular associated EIIIB FN and FN V domain may play a role in corneal wound healing.
Espaillat
A
A
Rhoads Molecular Immunology Laboratory, Harvard Medical School, Boston, MA.
Lee
S J
SJ
Arrunategui-Correa
V
V
Foster
C S
CS
Vitale
A
A
DiMeo
S
S
Colvin
R B
RB
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D003315:Cornea / Q000378:metabolism*; D065306:Corneal Injuries; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004848:Epithelium / Q000293:injuries / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014945:Wound Healing
D017931:DNA Primers; D005353:Fibronectins; D012333:RNA, Messenger