Polynucleotide constructs and uses thereof
A polynucleotide and nucleotide technology, applied in the field of polynucleotide constructs, can solve the problems of transcription level and hypoxia-inducible ratio disappointment
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Embodiment 21
[0051] Example 21 provides another example of HRE modification to enhance its hypoxic response.
[0052] A HRE may also comprise additional sequences, such as those with which it is naturally associated as part of an enhancer, or other sequences.
[0053] The expression level of the NOI or NOIs under the regulation of the specific polynucleotides of the present invention can be adjusted by manipulating the enhancer / promoter region. For example, different regions within a promoter region have different gene regulatory activities. The role of these different regions can often be assessed using vector constructs containing different variants of the promoter missing specific regions, (ie deletion analysis methods). This method can be used to determine, for example, the smallest region that confers tissue specificity or hypoxia inducibility.
[0054] Preferred promoters for use in the polynucleotides of the present invention are strong promoters such as viral promoters. For exam...
Embodiment 1
[0299] Example 1 - Construction of Optimal Hypoxia Response Element - Obhre11: A Mouse PGK HRE-Based Promoter
[0300] A variety of HRE constructs are known in the art. However, as mentioned above, to date, the degree of hypoxia-restricted expression directed by these sequences in constructs has not been entirely satisfactory. Appropriate HRE constructs should be able to direct high-level expression in a hypoxia-restricted manner. We therefore tested the hypoxia-specific activity of several different constructs, including two novel constructs based on three murine PGK HRE direct repeats cloned upstream of the SV40 promoter sequence, in the hope of obtaining a A sequence of desired properties.
[0301] Materials and Methods
[0302] Plasmid construction
[0303] Synthetic oligonucleotides containing the hypoxia response element (HRE) sequence were synthesized and cloned as a Bg1II / BamH1 fragment into the BamH1 site of the pGL3 promoter plasmid (Promega; Genbank accession no...
Embodiment 2
[0344] Example 2 - PGK HRE containing the transcription factor HIF-1 consensus sequence within OBhre11 can elicit hypoxia-mediated expression in macrophages, a cell that does not express HIF-1.
[0345] All of the HRE promoters described above contain the DNA motifs required for the binding of canonical HREs to the transcription factor HIF-1. This is a heterodimer composed of HIF 1α and HIF 1β, which belongs to the basic helix-loop-helix (bHLH) / PAS domain protein family (the original members of this gene family are: Period, ARNT and SIM). HIF 1α was originally identified as a protein capable of binding to the HRE of the erythropoietin gene in hepatoma cells (Wang et al., 1995), but it was later found to be involved in the regulation of other genes regulated by hypoxia in most cells. regulation of gene families.
[0346] Macrophages are able to infiltrate tumors to the extent that they constitute a substantial proportion of the total tumor mass. In particular macrophages can ...
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