Difference between revisions of "Part:BBa K4585012"
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Revision as of 16:14, 6 October 2023
pcDNA3.1(+)-3XHA-GAL4-VP64-NLS
The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid, which could express GAL4-VP64, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). VP64 is a transcription factor that, when used in combination with GAL4, can activate UAS and initiate the expression of downstream genes.
pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid was obtained through homologous recombination of the VP64 homologous recombination insert (BBa_K4585002) with pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector (BBa_K4585006). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.
1 Pattern Diagram
Fig.1 The model diagram of pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
2 Experiment
2.1 Method
The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid could express GAL4-VP64, thereby activating 9×UAS, which could activate the expression of its downstream gene, GAL4-KRAB or Luciferase.
2.2 Results
HEK 293T cells were transiently transfected with GAL-VP64 and GAL-KRAB plasmids, and an appropriate amount of Luciferase plasmids were transfected to simulate GnRH. The experiment showed that the GAL-VP64 plasmid could initiate the expression of GAL4-KRAB and Luciferase.
Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng
3.Caution
After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.