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

We based on the sequence of the pcDNA3.1 (+) plasmid.The Gal 4 sequence, 3×HA sequence were linked into the pcDNA3.1 (+) plasmid with the help of homologous recombination experiments. Special primers were designed and then the plasmid was cut into linear sequences of about 5600bp by PCR.The two ends of this linear sequence can be complementary to the two ends of the KRAB homologous recombination fragment , and the purpose is to conduct homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3 HA-Gal 4-KRAB-NLS plasmid.

1 Pattern Diagram

Fig.1 The agarose gel electrophoresis of pcDNA3.1(+)-3×HA-Gal4-KRAB-NLS linearized vector

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℃.