Chimeric gene for improving physiological cyp3a4 expression of cultured human hepatocytes in vitro and construction method thereof
A technology of chimeric gene and in vitro culture, applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of not reaching the expression level of primary hepatocytes, and achieve the effect of strong control
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Embodiment 1
[0032] Construct recombinant eukaryotic expression vectors PCI-hPXR-P53 and PCI-P53-hPXR, the steps are as follows:
[0033] 1. According to the PXR and wild-type P53-AD gene sequences included in the GenBank database, design specific primers, and use PCR method to clone PXR and wild-type P53-AD gene sequences from cDNA derived from normal human primary liver cells;
[0034] 2. Through restriction endonuclease sites, the PXR and wild-type P53-AD genes were directional cloned into the eukaryotic expression vector PCI-neo (such as figure 1 shown), the recombinant eukaryotic expression vector PCI-hPXR-P53 was screened and identified by enzyme digestion, sequencing and other methods, such as figure 2 shown;
[0035] 3. Through restriction endonuclease sites, PXR and wild-type P53-AD genes were directional cloned into the eukaryotic expression vector PCI-neo (such as figure 1 shown), the recombinant eukaryotic expression vector PCI-P53-hPXR was screened and identified by enzyme ...
Embodiment 2
[0037] The dual-luciferase reporter gene system is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase (fireflyluciferase). Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation of luciferin, it will emit bioluminescence. Combined with the co-reporter gene of marine coelenterate luciferase, when using firefly luciferase to quantify gene expression, marine cavity Enteroluciferase to reduce experimental variability. The bioluminescence released during luciferin oxidation can then be measured by a fluorometer, also known as a luminometer or a liquid scintillation meter. Luciferin and luciferase, a bioluminescent system, can detect gene expression extremely sensitively and efficiently.
[0038] A transcription factor is a protein molecule with a special structure that regulates gene expression, also known as a trans-acting factor. Some transcription factors only bind to specific sequences in their t...
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