Overview
The "GnRH Timer" we designed is a mammalian cell expression system that can secrete GnRH in a period of 2 to 5 hours. In our current experiment, the best experimental results have successfully adjusted the GnRH period to 8.16 hours.We have successfully shortened the period using the following three methods: ① Adjusting the overall action levels of GAL4-VP64 and GAL4-KRAB plasmids; ② Adjusting the dosage ratio of GAL4-VP64 and GAL4-KRAB plasmids; ③ Changing the half-life of GAL4-VP64 and GAL4-KRAB to accelerate their degradation rate.But there is still a certain gap between the experimental results and the expected 2-5 hours.In the future, we will continue to try to shorten the period of GnRH, and there are currently several envisioned improvement methods.But there is still a certain gap between the experimental results and the expected 2-5 hours. In the future, we will continue to try to shorten the cycle of GnRH, and there are currently several envisioned improvement methods.
Further Design
Shortening the expression period of GnRH
A.Repeat the experiment in ① and ② above using GAL4-VP64-Degron and GAL4-KRAB Degron plasmids connected to Degron, further shortening the period of GnRH.
B.Attempt to connect multiple Degron protein degradation sequences in GAL4-VP64 and GAL4-KRAB plasmids to enhance degradation rate, further reduce the time for transcription factors to function, and shorten the period of GnRH.
C.Change the repetition rate of UAS, regulate the effect of transcription factors, and shorten the period of GnRH.
Detecting the secretion cycle of GnRH
In existing experiments, in order to facilitate the detection of GnRH secretion, we used Luciferase instead of GnRH in 293T cells.In order to more accurately detect the actual secretion cycle of GnRH, we plan to co transfect AEQ, GnRH, GAL4-VP64, GAL4-KRAB plasmids into 293T cells, and take the supernatant secreted by the cells at fixed intervals for calcium ion flow detection experiments.After measuring the data, the standard curve can be used to obtain the secretion of GnRH, thereby determining whether GnRH exhibits rhythmic secretion.
Adjusting the appropriate amount of GnRH action
The GnRH secretion data obtained from 2.2 can also help us determine whether the content of GnRH secreted by the engineered cell meets the needs of the human body, and assist us in determining the specific transfection amount of the plasmid when preparing the engineered cell.
