FIGURE 2. Demonstration of stable integration of shGrb2 into the COS7 genome.

A, PCR amplification of pU6⁺²⁷-shGrb2 and pU6⁺²⁷-shControl markers (Neo^R and shRNA insert) in COS7^shGrb2 and COS7^shControl cells, respectively. After eight cellular passages in the continuous presence of 0.5 mg/ml neomycin, genomic DNA from COS7^shGrb2 and COS7^shControl cells were obtained and amplified by PCR in order to detect the neomycin resistance gene (Neo^R: 513 bp) or each of the inserts encoded in the shRNA inserts (shGrb2: 706 bp or shControl: 657 bp). The PCR fragments of 706 bp (the shGrb2 insert) and 657 bp (the shControl insert) obtained from COS7^shGrb2 and COS7^shControl cells, respectively, were sequenced in both directions using the same PCR primers used for PCR amplification (not shown). B, As controls, the Neo^R gene encoded exclusively in the pU6⁺²⁷ plasmids was also PCR-amplified from COS7^shGrb2, COS7^shControl and COS7^WT genomic DNA, as well as directly from the purified plasmids pU6⁺²⁷-shGrb2 or pU6⁺²⁷-shControl.