Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells

Abstract

The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure

“…HUVEC cDNA library screening was performed as it was previously described [Csortos et al, 1999]. Briefly, 10 6 clones of a random hexamer and oligo dT primed HUVEC λgt 11 cDNA library (kindly provided by Dr. David Ginsburg, Ann Arbor, USA) were screened.…”