1. A Novel Adaptor Protein Orchestrates Receptor Patterning and Cytoskeletal Polarity in T-Cell Contacts
2. Hybridization with mouse CD2AP cDNA was used to clone a genomic fragment from a 129/SvJ phage library. A 6-kb fragment 5′ of the exon encoding the first SH3 domain was generated by polymerase chain reaction (PCR) with the following primers: 5′-GCGGCCGCTGTATATGATATGAGTACACTGTAG-3′ and 5′-GCGGCCGCACATTCATTACATACTGCATTC3′. After digestion with Not I it was cloned into the Not I site of the targeting vector pTK.NEO.UMS [
3. Reis L. F., Ruffner H., Stark G., Aguet M., Weissman C., EMBO J. 13, 4798 (1994);
4. ]. A 1.1-kb fragment 3′ of the exon encoding the first SH3 domain was generated with the two primers 5′-TAGAACATCGATGTCAAGAAATAAATGCATATG-3′ and 5′-AATCTAATCGATTCCCAGCATCCACAGCTC-3′ and cloned into the Cla I site of the targeting vector.
5. Transfection of RW-4 embryonic stem (ES) cells was performed by the Washington University ES Cell Core. Homologous recombinants were screened by Southern blotting with a 500–base pair fragment (encompassing the Bam HI–Kpn I fragment) generated with the primers 5′-GGATCCCCTGGAGCTGTGGATG-3′ and 5′-GGTACCATTTCCATTTCTGCTAGG-3′ which is external to the 3′ arm of the targeting vector. Three positive clones were identified and two clones were microinjected into C57/Bl6 blastocysts with standard methods [