Method for the construction of specific promoters
The present application relates to a system for designing promoters for selective expression of genes. Thereby identified transcription regulatory elements are selected according to a specific methodology and used to create a library of transcription regulatory elements, which are then used to construct specific promoters, especially tissue-specific promoters.
1. A method for making a transcription-enhancing combined promoter cassette, the method comprising:
(a) identifying a plurality of transcription factor regulatory elements (TFREs), wherein each of the plurality of TFREs is associated with a plurality of genes, each of which is differentially expressed in a particular cell type or tissue type, or under a particular condition;
(b) determining the frequency, length and SYN value for each of said plurality of TFREs provided in step (a), and selecting TFREs from among the plurality of TFREs provided in step (a), wherein each of the selected TFREs (1) is within 20 kilobases of more than fifty percent of the plurality of genes defined in step (a), and (2) has a SYN value larger than 0.3, wherein the SYN value of a TFRE is defined as frequency (1/length) , wherein frequency is defined as the number of occurrences of a given TFRE being associated with any of the plurality of genes, divided by the total number of all TFREs associated with any of the plurality of genes, and wherein the frequency is its frequency within 20 kilobases of any of the plurality of the genes and length is the length in nucleotides of the TFRE;
(c) constructing a library of randomly combined elements by randomly combining TFREs selected in step (b); and
(d) inserting combined elements from the library of (c) in a vector with a minimum promoter and a reporter gene, thereby generating a transcription-enhancing combined promoter cassette.
2. The method of claim 1 , further comprising step (e): inserting the vector into a host cell.
3. The method of claim 2 , wherein step (e) produces a plurality of host cells and the method further comprises step (f): screening for a host cell produced in step (e) that shows enhanced expression of the reporter gene.
4. The method of claim 3 , further comprising identifying the combined promoter cassette in the host cell produced in step (e) or the host cell selected in step (f).
5. The method of claim 1 , wherein the library of randomly combined selected TFREs in step (c) is made by mixing individual double stranded DNA sequence elements encoding at least the selected TFREs together under ligation reaction conditions.
6. The method of claim 1 , wherein the reported gene in step (d) is LacZ or GFP.
7. The method of claim 1 , wherein each of the plurality of genes is differentially expressed under the particular condition, which is a disease condition.
8. The method of claim 7 , wherein the disease condition is cancer.
9. The method of claim 1 , wherein each of the plurality of genes is differentially expressed under the particular condition, which is exposure to a particular biological agent, chemical agent, or microbial pathogen.
10. The method of claim 1 , wherein a plurality of combined promoter cassettes are generated in step (d) and the method further comprises selecting one or more of the combined promoter cassettes so produced, wherein:
the number of TFREs per promoter in each of the selected combined promoter cassettes is greater than the average number of TFREs per promoter for the plurality of combined promoter cassettes generated in step (d); or
the number of TRFEs per promoter in each of the selected combined promoter cassettes is greater than 2.
11. The method of claim 1 , wherein the frequency of occurrence of the TFRE within 20 kilobases of any of the plurality of the genes is the frequency of occurrence of the TFRE within 20 kilobases in the sense strand of any of the plurality of the genes.