Method

A vector comprising a nucleotide sequence for use in a method of screening multiple putative gene regulatory element (pGRE) in parallel, comprising the nucleotide blocks, in sequence PRIMREG1-pGRE-PRIMREG2-REPSEQ-IRES-SSR, or GENE-PRIMREG1-pGRE-PRIMREG2

• wherein:
• PRIMREG1 is either a primer recognition sequence 1, or a FLEx cassette comprising a primer recognition sequence 1.
• pGRE is a putative gene regulatory element,
• PRIMREG2 is either a primer recognition sequence 2, or a FLEx cassette comprising a primer recognition sequence 2,
• GENE comprises nucleotide blocks REPSEQ, IRES and SSR, wherein
• REPSEQ is a reporter gene and is either present or absent,
• IRES is either an internal ribosome entry site or a 2A self-cleaving peptide, and
• SSR is a site specific recombinase gene;
• wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

A method of screening multiple putative gene regulatory elements (pGREs) in parallel comprising the steps:

• a. generating or having generated a population of vectors comprising a nucleotide construct comprising nucleotide blocks, in sequence PRIMREG1-pGRE-PRIMREG2-GENE, or GENE-PRIMREG1-pGRE-PRIMREG2
• wherein:
• PRIMREG1 is either a primer recognition sequence 1, or a FLEx cassette comprising a primer recognition sequence 1.
• pGRE is a putative gene regulatory element,
• PRIMREG2 is either a primer recognition sequence 2, or a FLEx cassette comprising a primer recognition sequence 2,
• GENE comprises nucleotide blocks REPSEQ IRES and SSR, wherein
• REPSEQ is a reporter gene,
• IRES is either an internal ribosome entry site or a 2A self-cleaving peptide, and
• SSR is a site specific recombinase gene;
• wherein at least one of PRIMREG1 and PRIMREG2 is a FLEx cassette comprising a primer recognition sequence;
• b. contacting target cells with the population of AAV vectors under conditions suitable for transduction of the target cells;
• c. isolating DNA from the transduced target cells;
• d. selectively amplifying active and/or inactive pGREs using PCR, and
• e. determining the sequence of active and/or inactive pGREs.