PMC:2575619 / 8677-11282
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2575619","sourcedb":"PMC","sourceid":"2575619","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2575619","text":"RT-PCR cloning of novel alternative splice variants and generation of alternative splice variant adenoviruses\nmRNAs that represent major human tissue types from healthy or diseased tissues and from cell lines were purchased from Clontech (Mountain View, CA, USA) and from Strategene (La Jolla, CA, USA), and were pooled. Synthesis of the first-strand cDNA was performed using STRATASCRIPT reverse transcriptase (Stratagene) following the manufacturer's instructions. For PCR amplification, gene-specific PCR primers were selected. The forward primers flanked the start codon. The reverse primers were selected from the transmembrane region of the receptors. PCR conditions were 35 cycles of 95°C for 45 seconds, 60°C for 50 seconds, and 72°C for 5 minutes. The reaction was terminated with an elongation step of 72°C for 10 minutes.\nPCR products were electrophoresed on 1% agarose gel, and were stained with Gelstar (BioWhittaker, Walkersville, MD, USA). The DNA bands were extracted with the QiaQuick® gel extraction kit (Qiagen), ligated into the pDrive UA-cloning vector (Qiagen), and transformed into Escherichia coli. Recombinant plasmids were selected on bacterial agar plates containing 100 μg/ml carbenicillin. For each transfection, 200 to 1,000 colonies were randomly picked and their cDNA insert sizes were determined by PCR with M13 forward vector and reverse vector primers. Representative clones from PCR products with distinguishable molecular masses as visualized by fluorescence imaging (Alpha Innotech, San Leandro, CA, USA) were completely sequenced.\nFor the bioinformatics analyses, computational analysis of alternative splicing was performed by alignment of each cDNA sequence to its respective genomic sequence using SIM4 (software for analysis of splice variants; Pennsylvania State University, Centre County, Pennsylvania, USA). Only transcripts with canonical (for example, GT–AG) donor–acceptor splicing sites were considered for further analysis.\nThe replication-deficient adenoviral expression system ViraPower was used for subcloning and expression of the ASV proteins following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Recombinant ASV-expressing adenoviruses were produced and amplified in HEK293A cells (Invitrogen), purified through a double-cesium chloride centrifugation procedure, and titrated by measuring the plaque-forming units or the infectious particle units in HEK293 cells. The Adv-Fc control virus, expressing a murine IgG2a Fc fragment, has been previously described [44]. Adv-LacZ virus was purchased from Welgen (Worcester, MA, USA).","divisions":[{"label":"Title","span":{"begin":0,"end":109}}],"tracks":[{"project":"2_test","denotations":[{"id":"18593464-11274374-4200938","span":{"begin":2538,"end":2540},"obj":"11274374"}],"attributes":[{"subj":"18593464-11274374-4200938","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecaf93","default":true}]}]}}