Part:BBa_K4585013

pGL4.35-3XHA-9XGAL4UAS-KRAB-NLS

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, which could express GAL4-KRAB, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). KRAB is a transcription factor that represses downstream gene expression when combined with GAL4.

pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid was obtained through homologous recombination of the KRAB homologous recombination insert (BBa_K4585003) with pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector (BBa_K4585008). 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 pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS

2.1 Method

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid could express GAL4-KRAB, and GAL4-KRAB could bind 9×UAS and inhibit downstream gene expression, therefore GAL4-KRAB could inhibit GnRH expression. Considering that the plasmid itself contains a 9×UAS sequence, GAL4-KRAB can also suppress its own expression.

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℃.