How do bicistronic (IRES) vectors express two proteins simultaneously?

Bicistronic lentiviral vectors contain an optimized internal ribosome entry site (IRES) that permits your gene of interest and a second gene (This is often the gene for a fluorescent protein or drug selection marker) to be coexpressed from a single mRNA transcript. Although translation initiation occurs almost exclusively at the 5' cap of eukaryotic mRNAs, the IRES attracts ribosomes to begin [...]

Do I have to include an internal poly(A) signal in the retroviral expression cassette for my gene of interest (GOI)?

No. The GOI or selection marker expression cassette in the retroviral vector should not include a poly(A) signal or any other transcription termination signal, since it is located in the 3' LTR of the retroviral vector. A transcription termination sequence in the middle of a retroviral construct can lead to the premature cleavage of viral genomic mRNA, resulting in the loss of downstream [...]

What fraction of the wild-type MMLV genome is present in Takara Bio’s retroviral vectors?

Approximately 12–17%, depending on the vector backbone. pLXRN contains 11.9% of the wild-type MMLV genome, and the MSCV vectors contain 16.9% of the wild-type MMLV genome.Retrovirus [...]

What is the cloning/packaging capacity of a retroviral vector from Takara Bio?

Wild-type moloney murine leukemia virus (MMLV) contains ~8.2 kb of genome, including both LTRs. Artificially creating a genome larger than 8.2 kb will result in unstable viral particles and a dramatic drop in viral titer. For recombinant retroviruses such as those generated using Retro-X systems, much of the viral genome has been replaced with other useful sequences such as selection markers or [...]

What are the most common artifacts of cDNA synthesis with SMARTer kits?

Elevated baseline in the Bioanalyzer trace. This is commonly due to the presence of SPRI beads in the cDNA preparation. Although SPRI beads themselves do not fluoresce (nor will they bind the dye included in the Agilent High Sensitivity DNA Kit), any DNA remaining on the bead will bind dye and fluoresce. To prevent bead-carryover: Leave the sample on the magnetic stand for an additional five [...]