Ps gfpneo

RT-PCR was used for cloning of PRG3 from rat, mouse and human mRNA samples. For sequence alignments and homology searches of PRGs we utilized the www.ncbi.nlm.nih.gov database and A Plasmid editor software (ApE; MW Davis, Utah, USA). Trans-membrane domains have been predicted using the Kyte Doolittle algorithm and all orthologous sequences of PRG3 (human, mouse and rat) are deposited at the NCBI database (Human PRG3 GenBank accession no. AY304516; Rattus norvegicus PRG3 GenBank accession no. AY299399; Mus musculus PRG3 GenBank Accession no. AY345342). We amplified the fragments by PCR and cloned the resulting amplicons into the pEGFP (Takara, Heidelberg, Germany), and pCLEG (Stefan Schumacher, Ulm, Germany) vectors. C-terminal domain of PRG3 was cloned respectively. According to the criteria of Naito et al [51 (link)] two short interfering RasGRF1 RNAs were designed. Cloning of the synthetic oligonucleotides into the pSuperRFP vector (modified pS-GFPneo; OligoEngine, Seattle, USA) was performed by digesting the empty vector with Bgl2 and EcoR1 according to the manufacturer's instruction.

Reverse transcription-polymerase chain reaction was used for full length cloning of PRG3 from rat, mouse and human mRNA samples as described previously [13 (link)]. For sequence alignments and homology searches of PRGs we utilized the www.ncbi.nlm.nih.gov database and A Plasmid editor software (ApE; MW Davis, Utah, USA). Transmembrane domains have been predicted using the Kyte Doolittle algorithm and all orthologous sequences of PRG3 (human, mouse and rat) are deposited at the NCBI database (Human PRG3 GenBank accession no. AY304516; Rattus norvegicus PRG3 GenBank accession no. AY299399; Mus musculus PRG3 GenBank Accession no. AY345342). For construct cloning we cloned fragments by PCR and inserted the resulting amplicons into the pEGFP (Takara, Heidelberg, Germany), and pmRFP (Kes Jalink, NKI, Amsterdam, the Netherlands) vectors. C-terminal domain of PRG3 was cloned by PCR amplification out of the full length clone and inserted the amplicon into linearized pEGFP vectors. According to the criteria of Naito et al. [40 (link)] three 19-mer short interfering RNAs were chosen for RNA interference with rodent PRG3 transcripts (GenBank acc. AY299399). Cloning of the synthetic oligonucleotids into the pSuperGFP vector (pS-GFPneo; OligoEngine, Seattle, USA) was performed by digesting the empty vector with Bgl2 and EcoR1 according to the manufacturer's instruction.