Mammalian Selection：G418, neo

Improved System

The improved LacSwitch? II inducible mammalian expression system consists of 3 vectors: pOPI3CAT vector from the original LacSwitch system and two new vectors, pCMVLacI and pOPRSVI/MCS, that dramatically improve the system’s performance and versatility. Lac repressor protein, produced from the pCMVLacI vector, blocks transcription by binding the Lac repressor protein to a specific DNA sequence （the operator） in the pOPI3CAT and pOPRSVI/MCS vectors. IPTG, which has no adverse effect in eukaryotic cells, decreases the binding affinity of the Lac repressor protein to the operator sequences, triggering transcription and expression of the inserted gene. The LacSwitch II system allows induction of the gene of interest within 4-8 hours.

Tight Repression

The new pCMVLacI vector replaces the p3’SS vector from the original system. The pCMVLacI vector provides greater repression of the gene of interest due to the increased strength of the CMV promoter. High expression of the lacI gene provides 2- to 10-fold tighter repression of the gene of interest than the original p3’SS vector. A nuclear localization sequence （NLS） directs expression of Lac repressor to the nucleus.

Directional Insertion of the Gene of Interest

To facilitate directional cloning, the multiple cloning site from pBluescript? II SK was inserted into pOPRSVI. The resulting pOPRSVI/MCS vector permits directional insertion of the gene of interest, decreasing the time required for screening. The presence of T3 and T7 promoters on either end of the MCS in the pOPRSVI/MCS vector allows easy sequencing and verification of positive inserts using T3 and T7 primers.

Removal of the RSV Promoter

The pOPI3CAT operator vector is included in the LacSwitch II system for replacing the RSV-LTR promoter with a promoter of interest. This vector contains three operators in the intron and unique restriction sites for removal of the RSV promoter.