Q: Which expression vector backbone should I use?
A: The retroviral expression vector will depend on the target cells being used. The pMCs vectors are used for ES and EC cells, the pMYs vectors are used with hematopoietic stem cells and progenitors, and the pMXs and pBABE vectors are suitable for general gene expression. The pMYs and pMCs vectors have modified LTRs that allow for gene expression in their respective cell types.
Q: What is the sequencing primer you suggest for your Retroviral Expression Vectors?
A: This is the sequencing primer we use for our retrovirus expression vectors: GACGGCATCGCAGCTTGGATACAC.
Q: Which E.coli strain should be used for amplification of the Retroviral Expression Vectors?
A: We recommend using Invitrogen Stbl3 competent cells for plasmid amplification, which will minimize recombination that can happen through the viral LTR sequences. If cells other than Stbl3 were used, such as DH5a or Top10, a restriction digest can be performed to confirm that the plasmid has maintained the original size.
Q: Does my gene of interest need to be in frame?
A: To clone your gene into a retroviral expression vector, you will need the entire coding region of the gene, from start to stop codon. You do not need to worry about the frame of your insert; it is similar to any cloning vector where translation will start with the ATG of your cloned sequence and continue to the stop codon.
Q: Should the insert contain a polyA site?
A: Inserts cloned into retroviral expression vectors should not contain polyadenylation signals. The viral 3’ LTR contains a polyA site, so the presence of polyadenylation signals upstream may result in premature cleavage of the viral genome during transcription. This would affect viral genome packaging and result in low titer.
Q: Can retroviral expression vectors be used for transient transfection experiments?
A: Our retroviral reporter constructs may be used for a transient transfection of mammalian cells, without packaging the virus.