Strategy for the high-throughput in vivo assay. (A) Design of the gene-specific forward and reverse primers. The two common sequences Tag1 and Tag2 are used as margins to connect the cDNA with other DNA fragments. (B) Sample preparation. The gene-specific forward and reverse primers in (A) were used to amplify each targeted CDS. Red and green boxes are the two common sequences produced by Tag1 and Tag2 during PCR. The DNA fragments for CMV-TIP-1-TNNC2 and SV40 were obtained from the pACT vector. The PCR products were connected with the DNA fragments for CMV-TIP-1-TNNC2 and SV40 using FPCMV5 and LGT10L primers (ACT sample). (C) BIND-construct preparation. The DNA fragment for CMV-GAL4 was amplified from the pBIND vector using FPCMV6 and RPCMVGAL4 primers. A region of 20amino acids at the C-terminus of Rhotekin molecule was mediated and connected to the DNA fragments for CMV-GAL4 and SV40 (BIND construct).
Hoat et al. BMC Cell Biology 2009 10:69 doi:10.1186/1471-2121-10-69