Part:BBa_K4585007

pcDNA3.1(+)-3xHA-Gal4-KRAB-NLS linearized vector

The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3xHA-Gal 4-KRAB-NLS plasmid.

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

2.1 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℃.