Inducible expression systems
AssigneeTET Systems GmbH & Co. KG
InventorsBenjamin Berkhout, Atze T. Das
Life of this patent
Estimating the term…
Abstract
Provided is an rtTA and single chain rtTA variants and uses thereof for inducible expression of a nucleic acid of interest. Nucleic acid molecules comprising an improved rtTA and/or sc rtTA sequence according to the invention are also provided, as well as vectors, replicons and cells comprising such nucleic acid molecules.
Claims83
A method for inducibly expressing a nucleic acid sequence of interest, the method comprising:
- providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system that comprises a reverse tetracycline-controlled transactivator (rtTA) encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 9, and/or 19, and/or 37, and/or 56, and/or 67, and/or 68, and/or 138, and/or 157, and/or 171, and/or 177, and/or 195;
- introducing said nucleic acid construct to a suitable expression system;
- and
- allowing for inducible expression of said nucleic acid sequence of interest.
A synthetic or recombinant nucleic acid sequence comprising a rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 9, and/or 19, and/or 37, and/or 56, and/or 67, and/or 68, and/or 138, and/or 157, and/or 171, and/or 177, and/or 195.
A synthetic or recombinant amino acid sequence comprising a rtTA sequence and/or a single chain rtTA sequence, which rtTA sequence and/or single chain rtTA sequence comprises an isoleucine at position 9, and/or an alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine at position 19, and/or a cysteine, methionine, glutamine, threonine, histidine, leucine or arginine at position 37, and/or a lysine or glutamine at position 56, and/or a serine at position 67, and/or an arginine at position 68, and/or a tyrosine at position 86, and/or an aspartate or serine at position 138, and/or a lysine at position 157, and/or a lysine at position 171, and/or a leucine at position 177, and/or a serine at position 195, and/or a threonine at position 209.
In a method of tetracycline-inducible and/or minocycline-inducible expression of a nucleic acid sequence of interest, the improvement comprising:
- utilizing the recombinant nucleic acid sequence comprising an rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutation or a combination of mutations as depicted in FIG. 15, except for the wild type rtTA and the F86Y A209T variant, for tetracycline-inducible and/or minocycline-inducible expression of a nucleic acid of interest.
A method for inducibly expressing a nucleic acid sequence of interest, the method comprising the steps of:
- providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system that comprises a reverse tetracycline-controlled transactivator (rtTA) encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 67, optionally with one or more additional mutations in a codon at rtTA amino acid position 9, 12, 19, 37, 56, 68, 86, 138, 157, 171, 177, 195 or 209;
- introducing said nucleic acid construct to a suitable expression system;
- and
- allowing for inducible expression of said nucleic acid sequence of interest.
A synthetic or recombinant nucleic acid sequence comprising a rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 67, optionally with one or more additional mutations in a codon at rtTA amino acid position 9, 12, 19, 37, 56, 68, 86, 138, 157, 171, 177, 195 or 209.
A transactivator, having the DNA sequence (5′- 3′ orientation):
- (SEQ ID NO:
- 28)
- atgtctagactggacaagagcaaagtcataaactctgctctggaattact
- caatggagtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagc
- tgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggcc
- ctgctcgatgccctgccaatcgagatgctggacaggcatcatacccactc
- ctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgcca
- agtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcat
- ctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagct
- cgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctc
- tgtccgccgtgggccactttacactgggctgcgtattggaggaacaggag
- catcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcc
- cccacttctgaaacaagcaattgagctgttcgaccggcagggagccgaac
- ctgccttccttttcggcctggaactaatcatatgtggcctggagaaacag
- ctaaagtgcgaaagcggcgggccgaccgacgeccttgacgattttgactt
- agacatgctcccagccgatgcccttgacgactttgaccttgatatgctgc
- ctgctgacgctcttgacgattttgaccttgacatgctccccgggtaa.
A transactivator, having the amino acid sequence:
- MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEML DRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFL CQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQ GAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG*(SEQ ID NO:
- 29).
A method for inducibly expressing a nucleic acid sequence of interest, the method comprising the steps of:
- providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system that comprises a reverse tetracycline-controlled transactivator (rtTA) encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 67;
- introducing said nucleic acid construct to a suitable expression system;
- and
- allowing for inducible expression of said nucleic acid sequence of interest.
A synthetic or recombinant nucleic acid sequence comprising a rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 67.
Dependencies are inferred from the claim text.