Cre-lox based gene knockdown constructs and methods of use thereof
The present invention relates to vectors, compositions and methods for conditional, Cre-lox regulated, RNA interference. The vectors allow for spatial and temporal control of miRNA expression in vivo.
1. A vector comprising
a. a first pair of loxP sequences inverted in orientation with respect to each other,
b. a nucleic acid cassette comprising a first nucleic acid encoding
(i) at least one nucleotide sequence that can form a hairpin structure comprising at least one miRNA agent, and
(ii) miRNA sequences flanking the at least one nucleotide sequence, wherein the miRNA sequences flanking the at least one nucleotide sequence are derived from endogenous miRNA sequences that flank wild-type hairpin structures, and
wherein the nucleic acid cassette is positioned in an antisense orientation between the first pair of loxP sequences,
c. a second pair of loxP sequences distinct from the first pair of the loxP sequences, inverted in orientation with respect to each other, wherein the first pair of loxP sequences and the second pair of loxP sequences are configured such that flipping of the first pair results in excision of the sequences between the second pair.
2. The vector of claim 1 , wherein the vector further comprises a second nucleic acid encoding a selectable marker in sense orientation, wherein the second nucleic acid is positioned between the first pair of loxP sequences.
3. The vector of claim of claim 2 , wherein the nucleic acid cassette is 3′ with regard to the second nucleic acid, and wherein a first loxP sequence of the second pair of loxP sequences is positioned between the second nucleic acid and the nucleic acid cassette, and a second loxP sequence of the second pair is positioned 3′ with respect to the first pair of loxP sequences.
4. The vector of claim 3 , wherein the nucleic acid cassette further comprises a third nucleic acid encoding a second selectable marker, wherein the third nucleic acid is in antisense orientation and fused in frame to the first nucleic acid sequence.
5. The vector of claim 1 , wherein the first pair of loxP sequences comprises wild-type sequence.
6. The vector of claim 1 , wherein the second pair of loxP sequences comprises a mutated loxP.
7. The vector of claim 1 , wherein the first pair of loxP sequences comprises a loxP 5171 sequence.
8. The vector of claim 1 , wherein the second pair of loxP sequences comprises a loxP 2272 sequence.
9. The vector of claim 2 , wherein the second nucleic acid encodes two selectable markers fused in-frame with respect to each other.
10. A method of modulating gene expression in a cell, the method comprising:
introducing into the cell the vector of claim 1 ; and
inducing flipping of a first pair of loxP sequences contained within the vector thereby inverting a nucleic acid cassette contained within the vector from the antisense orientation to a sense orientation,
wherein the inversion of the nucleic acid cassette allows processing of at least one nucleotide sequence encoded by the nucleic acid cassette into at least one functional miRNA agent.
11. The method of claim 10 , further comprising:
inducing flipping of a second pair of loxP sequences contained within the vector.