Promoter engineering and genetic control
OwnerCurt Fischer, Gregory Stephanopoulos +2inventors
InventorsCurt Fischer, Gregory Stephanopoulos, Hal Alper, Elke Nevoigt
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Abstract
The present invention relates to expression vectors, wherein each vector comprises at least one gene of interest and a promoter operatively linked thereto wherein each promoter comprises a nucleic acid, whose sequence is randomly mutated with respect to that of the wild-type promoter and cells comprising the same. Methods utilizing either the vectors or cells of this invention, in optimizing regulation of gene expression, protein expression, or optimized gene or protein delivery are described.
Claims42
An isolated nucleic acid comprising a mutated DAN1 promoter corresponding to or homologous to SEQ ID No:
- 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
An isolated nucleic acid comprising a mutated DAN1 promoter corresponding to or homologous to SEQ ID No:
- 1, 2, 3, 4, 5, or 6.
An isolated nucleic acid comprising a mutated DAN1 promoter corresponding to or homologous to SEQ ID No:
- 1 or 2.
An isolated nucleic acid comprising a mutated DAN1 promoter, wherein said promoter comprises a mutation in at least one nucleic acid at one or more of the following positions of SEQ ID No:
- 11:
- a) 1-56
- b) 66-139
- c) 148-232
- d) 245-283
- e) 290-293
- f) 301-302
- g) 310
- h) 322-326
- i) 334-347
- j) 357-371
- k) 380-450 or
- l) 458-551.
An isolated nucleic acid comprising a mutated DAN1 promoter, wherein said mutated promoter has a sequence comprising a replacement of:
- a) a T with a C at nucleotide position 4, 15, 19, 36, 53, 56, 60, 66, 74, 75, 78, 86, 99, 132, 136, 176, 201, 205, 207, 216, 226, 228, 269, 277, 281, 285, 299, 303, 310, 327, 331, 332, 375, 376, 390, 428, 434, 467, 477, 480, 508, or 511;
- b) an A with a G at at nucleotide position 7, 18, 26, 40, 122, 135, 149, 153, 162, 164, 165, 171, 172, 187, 196, 211, 233, 234, 237, 241, 260, 274, 280, 308, 313, 322, 337, 343, 344, 346, 366, 368, 381, 384, 386, 396, 397, 402, 404, 422, 427, 429, 432, 445, 470, 490, or 492;
- c) a C with an A at nucleotide position 21;
- d) an A with a C at nucleotide position 237, 338, 469, 514, 518;
- e) a C with a T at nucleotide position 28, 296, 307, 373, 392, or 528;
- f) a G with an A at nucleotide position 22, 63, 391, or 439;
- g) a T with a G at nucleotide 198;
- or any combination thereof, of the sequence as set forth in SEQ ID NO:
- 11.
A method of determining optimized gene expression, wherein said gene is under the control of a regulatable promoter, said method comprising:
- a) Contacting a plurality of cells with a library of expression vectors, each vector comprising at least one gene of interest and a regulatable promoter operatively linked thereto,
- wherein each promoter comprises a nucleic acid, whose sequence is randomly mutated with respect to that of another in said library, and
- whereby relative changes in expression level of said gene of interest under conditions, which regulate gene expression, are a function of the mutation in said promoter sequence;
- b) Detecting gene expression levels of.cells in (a) cultured under said conditions, which regulate gene expression;
- and
- c) Identifying a cell from said plurality of cells in which expression levels under said conditions are optimized.
A method of regulating gene expression, said method comprising:
- a) Contacting a plurality of cells with a library of expression vectors, each vector comprising at least one gene of interest and a regulatable promoter operatively linked thereto,
- i) wherein each promoter comprises a nucleic acid, whose sequence is randomly mutated with respect to that of another in said library, and
- ii) whereby changes in an expression level of said gene, expression conditions of said gene of interest, or a combination thereof, of said gene of interest occur under regulatable conditions as a function of said mutation;
- b) Detecting gene expression in said plurality of cells obtained in (i), under conditions where wild-type gene expression occurs sub-optimally;
- c) Identifying a cell from said plurality of cells in which greater expression levels are obtained from said vectors, under conditions
- where wild-type gene expression occurs sub-optimally;
- and
- d) Culturing said cell identified in (c) under said conditions.
Dependencies are inferred from the claim text.