Genetic manipulation of GRB2 in T cells

For mammalian expression, HUT 78 T lymphocytes were transduced with pLK4 lentiviruses containing GRB2 shRNAs alone or with sequences encoding add-back wild-type or mutant GRB2 as previously described^{106 (link)}. Six silent mutations were introduced to the add-back GRB2 sequences to generate GRB2 messenger resistant to shRNA-mediated degradation. GRB2 derivatives bearing the mutations discussed were constructed by site-directed mutagenesis. The V123D putative monomer mutation was introduced by PCR using the following primers: 5′-GAAGTACTTCCTCTGGGTGGATAAGTTCAATTC-3′ and 5′-AGCTCATTCAAAGAATTGAACTTATCCACCCAGAG-3′. For the V122P/V123P putative dimer mutant, the primers were the following: 5′-CGGGAAGTACTTCCTCTGGCCGCCGAAGTTCAATT-3′ and 5′-GCTCATTCAAAGAATTGAACTTCGGCGGCCAGAGGAAG-3′. The N188D mutation was introduced by overlap extension PCR using the following primer pairs: 5′-CTTCAAGGTGCTCCGAGATGGAG-3′ and 5′-GGGTCTGAGTCATCCATGACATGGATAAAATCTC-3′; 5′-GTCATGGATGACTCAGACCCCAACTGGTG-3′ and 5′-GGGCGACCGGACTCTAGAG-3′. The N214D mutation was introduced using the following primers: 5′-GGACATAGAACAGGTGCCACAGCAG-3′ and 5′-AGTCGCGGCCGCTTAGACGTTCCGGTCCACGGGGGTGAC-3′. Primers were purchased from Integrated DNA Technologies (Coralville, IA). Expression of shRNA was under control of the U6 promoter, whereas add-back GRB2 expression was under control of the EF-1α promoter. Transduced cells were kept in selection with 1 μg/mL puromycin (Santa Cruz). For bacterial expression, the sequence encoding full-length, wild-type human GRB2 was cloned into the pET-28a(+) vector (Novagen, cat. no. 69864) and E. coli BL21 (DE3) cells transformed with the vectors as previously described^{36 (link)}. The same primers as listed above were used to generated Histidine-tagged variants of GRB2 for recombinant expression.