AAV with CMV7 promoter driven RFP-T2A-WGA-iCre

This AAV expresses RFP-T2A-WGA-iCre driven by an ubiquitous CMV7 promoter.

The CMV7 promoter is about 0.35 kb in length and is a shortened version of the commonly used CMV promoter. CMV7 has a truncated version of the CMV enhancer. When tested in several cell lines, this promoter provides an expression level that is about 3-5 fold lower than that of the original 0.6Kb CMV promoter.

The short 2A peptide sequences, when cloned in-frame between two genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. This differs from conventional approaches for multiple protein expressions, such as IRES-mediated bicistronic gene expression, which has several limitations including imbalanced protein expression. The use of 2A peptide sequences alleviates these concerns, since 2A-mediated “self-cleavage” gives rise to a 1:1 ratio of the two separate proteins. Several forms of 2A peptide are commonly used: T2A (Thoseaasigna virus 2A), P2A (porcine teschovirus-1 2A), E2A (equine rhinitis A virus), and F2A (foot-and-mouth disease virus, FMDV 2A).

Wheat germ agglutinin (WGA) is a transcellular tracer protein. It is commonly used in combination with another protein, such as Cre recombinase, to allow movement of the protein between cells

Cre is a recombinase from the P1 bacteriophage. This enzyme carries out site-specific recombination between two DNA recognition sites (LoxP sites) through a topoisomerase I-like mechanism. The Cre recombinase here has been codon-optimized leading to a higher level of Cre protein expression (iCre). The 34base pair (bp) loxP recognition site, ATAACTTCGTATA ATGTATGC TATACGAAGTTAT, consists of two 13 bp palindromic sequence which flank an 8 bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. For example, two separate DNA species both containing loxP sites can undergo fusion as the result of Cre-mediated recombination. DNA sequences found between two loxP sites on the same DNA molecule are said to be floxed. In this case, the products of Cre-mediated recombination depend upon the orientation of the loxP sites: DNA found between two loxP sites oriented in the same direction will be excised as a circular loop of DNA, whereas DNA between two loxP sites that are oriented oppositely will be inverted. Besides LoxP sites, other Lox sites have been designed and tested, such as Lox272 and LoxN etc. These Lox elements have been widely used for genetic studies.