Proteoglycans: Gene Cloning
Aggrecan is a large proteoglycan that plays roles in numerous tissues during vertebrate development and adult life. The 6,327-nt chick aggrecan coding sequence had been determined from overlapping clones, but a full-length cDNA, needed for use in transgenic expression studies, had not been constructed. The strategy employed to do so was to generate two overlapping cDNA subfragments that shared a unique restriction site in the overlap and then join them at that site. These subfragments were obtained and cloned into the TOPO-TA vector pCR2.1. Digestion of the two constructs with the shared-site enzyme, XbaI, produced vector/5′-cDNA and 3′-cDNA fragments with XbaI-ends; these were ligated to produce the final full-length cDNA.
Key wordsProteoglycan Aggrecan Cloning Strategy Full-length cDNA
The authors would like to thank Alex Baria and Danijela Maric for technical assistance.
This work was supported by R01 HD17732 (N.B.S.), and T32.G M 008720 and G M 007381 (M.C.).
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