When using the MACSelect Systems, do the transfected cells always express the gene of interest together with the selection marker?

The bicistronic pMACS 4-IRES.II vector contains a promoter followed by a multiple cloning site (MCS) for cloning the gene-of-interest without a polyA signal sequence. Downstream of the MCS are an internal ribosomal entry site (IRES) element and the truncated CD4 receptor. Both the gene of interest and the CD4 selection marker are transcribed into one bicistronic mRNA. The IRES element allows translation of the two open reading frames from this single, bicistronic mRNA into two separate proteins. Transfected cells that contain the pMACS 4-IRES.II vector with a correctly integrated gene of interest always express both the gene of interest and the CD4 selection marker. However, expression of the selection marker is often lower than expression of the protein of interest, ensuring that all selectable cells express a high amount of protein of interest.

The pMACS Kk.II vector contains one promoter followed by an MCS for cloning the gene of interest and a second promoter followed by the truncated H-2Kk gene. Both the gene of interest and the H-2Kk selection marker are transcribed and translated separately. Transfected cells containing the pMACS Kk.II vector with an integrated gene of interest will express both genes.

When using the pMACS Kk.II or the pMACS 4.1 vector for co-transfection, the selection marker gene and the gene of interest will be on separate vectors, so there is no guarantee that a transfected cell will contain both vectors. However, using an optimal co-transfection ratio will help increase the chance that most of the transfected cells containing the selection marker vector will also contain the vector with the gene of interest.

Generally, the type of cell used can affect the expression efficiency through promoter and IRES elements.