Ontent/1/1/DNA transformations A rat kidney cDNA library in the pcDNAI
Ontent/1/1/DNA transformations A rat kidney cDNA library in the pcDNAI T0901317 supplement vector [9] was purchased from Invitrogen (Carlsbad, CA). To increase the library transfection efficiency and maximize the integrity of the cDNAs, the library was digested in the vector sequence with the restriction enzyme SfiI and then religated. R4-7 cells in ten 10-cm dishes were cotransformed with 20 ug of the religated library DNA and 2 ug of pGKpuro plasmid by calcium phosphate-mediated transformation. Cells expressing transformed DNAs were selected by growth in culture medium containing 5 ug/ml puromycin. Each transformed cell was expected to receive about 2?0 different cDNAs. The cultures were expanded before the selections for virus susceptibility such that a pool of 105 cells contained about 2000 distinct puromycin resistant clones. Thus, there were about 50 sibling cells of each transformant in the pools at the time of selection for virus susceptibility. Retrovirus preparations Eco-neo or MuLV-N2 virus [20]; Eco-TK virus, and EcoGFP virus [21] were as previously described [6]. To generate Eco-His virus, the Neo resistance gene in the N2 vector was replaced with His resistance gene, and GP+E86 packaging cells [22] were stably transformed with the resulting vector DNA. Recipients were selected with histidinol and the resistant cells were pooled to generate Eco-His producer cells. Typical titers of the virus preparations on Rat2 cells were 107 cfu/ml for N2 virus; 2 ?104 for TK virus; 2 ?106 for Eco-His virus; and 105 cfu/ml for Eco-GFP virus. Viral transduction and selection Selections for virus sensitive cells were performed by infecting approximately 105 R4-7 cells per 10-cm dish in each round, with virus titers determined by infection of Rat2 cells. In the first round approximately 104 cfu of N2 virus were applied, and transductants were selected with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26780312 800 ug/ml G418. In the second round, approximately 2 ?103 cfu of Eco-TK virus were used, and transductants were selected with HAT medium (Gibco). In the third round, approximately 200 cfu of Eco-His virus were applied, and transductants were selected with medium containing 1 mg/ml histidinol. The multiplicities of all these infections with the selecting viruses were kept low, at less than 0.1 These low MOIs were required because infection of R4-7 cells at high MOI can override the block, perhaps by saturation of a titratable factor. Even at this low MOI, the presence of many siblings of each transformant implied that most cDNAs in the pool were tested for inducing virus susceptibility. Polymerase chain reactions cDNA inserts from the expression library were recovered from cell lines by PCR as follows. Genomic DNA was extracted (DNAeasy kit, Qiagen) and subjected to PCRwith primers hybridizing upstream from the CMV promoter (sequence 5′-GGGCCAGATATACGCGTT-3′) and downstream from the poly(A) addition region (sequence 5′-AATTTGTGATGCTAT-3′) of the pcDNAI vector. Conditions for the PCR were: ten cycles of 94 for 10 sec, 55 for 30 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25768400 sec, and 68 for 3 min, followed by 20 cycles of the same conditions but with an increase in the polymerase reaction time of 5 sec in each cycle. The amplified DNAs were cloned directly into the TOPO vector (Invitrogen) and used to transform DH10b bacteria to ampicillin resistance. DNAs were isolated from approximately fifty bacterial colonies for each original cell line. CAPER cDNAs were prepared from RC-2 mRNA preparations by standard RT-PCR methods using primers spanning the entire.