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Isolation of Wound-Specific cDNA Clones from a cDNA Library Prepared with mRNAs of Alkali-Burned Rabbit Corneas

Haseba Takeshi; Nakazawa, Mitsuru; Kao, Winston W-Y.; Murthy, Ramesh; Kao, Candace W-C.
Cornea: July 1991
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Alkali burn is one of the most severe corneal injuries. In order to gain a better understanding of the healing of alkali-burned corneas, it is necessary to identify and characterize proteins that are specifically synthesized by the injured corneal tissues. In this study, we developed a useful procedure to identify and isolate cDNA clones that encode messenger ribonucleic acids (mRNAs) that are specific and/or abundant in alkali-burned rabbit corneas (ARCs), but absent in normal rabbit corneas (NRCs). At first, a cDNA library was prepared by cloning cDNA of mRNA isolated from ARCs into the λORF-8 vector. A differential plaque hybridization was used to screen 2.5 x 104 plaque-forming units (pfu) from an ARC cDNA library using 32P-labeled cDNAs prepared from mRNA of ARCs and NRCs. Thirty-seven cDNA clones of mRNAs specific for ARCs were identified and isolated in their pureform. The cDNA inserts of these λORF-8 phages were subcloned into the pSM216 vector by in vivo recombination. The cDNA inserts then were characterized by restriction enzyme digestion, i.e., Bam HI, Hin dIII, and Eco RI. The size of the cDNA inserts ranged from 210 to 5,000 base pairs. Using Northern blot hybridization of total RNA prepared from polymorphonuclear neutrophils, mononuclear leukocytes, alkali-burned corneas, and normal corneas, the cDNA clones were divided into three groups. Five cDNA clones encoded mRNA of corneal cells in ARCs. Twenty-four cDNA clones derived from mRNA of inflammatory cells were present in alkali-burned corneas, but Northern blot hybridization failed to identify mRNA of discrete sizes. Eight cDNA clones encoded mRNA of inflammatory cells; the sizes of mRNAs ranged from 0.5 to 2 kilobase. Our strategy proved to be useful in isolating cDNA clones that are specific for alkali-burned corneas. Thus, further studies can be performed to examine roles of proteins that are encoded by the isolated cDNA clones.

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