At the beginning of the experiment designed by LZU-CHINA, the experimental direction
and purpose of NMU_China are very similar to ours. Therefore, a close long-term
cooperative relationship has been established between two teams. Our project uses
CRISPR-Cas13d technology to knock down the expression of ACE2 so as to inhibit
the entry of SARS-CoV-2 into cells to reduce viral load. There is something in
common with NMU_China's project to reduce viral load after virus infection. After
each other's project introduction, we established human practice, model, experimental
cooperation. The close communication and discussion between the two teams not only
benefited each other, but also increased the friendship between the two teams.
Part.1 Experimental improvement
NMU_China → LZU-CHINA
NMU_China sent LZU-CHINA HEK293T cell line: After LZU-CHINA and NMU_China cooperated
and discussed the experiment, they also carried out friendly mutual help. In the early
stage of LZU-CHINA cell experiment, team members found that there was a problem of cell
contamination in the process of cell culture in the process of subculture of HEK293T
cells. Under the condition that the culture method was correct, it was finally determined
that HEK293T cell source was contaminated by black glueworm. We have repeatedly
changed the HEK293T cell source in different laboratories, but it has always been
unable to solve the problem of black glueworm pollution. This setback also made our
experiment once stalled. After communication with the NMU_China team, it was known that
the NMU_China team also had HEK293T cell line to determine that there was no contamination
of black glueworm and that it could be subcultured continuously. So NMU_China sent
HEK293T cells by mailing to the LZU-CHINA team, and the LZU-CHINA team ensured that
subsequent cell experiments were carried out smoothly by replacing cell lines.
LZU-CHINA → NMU_China
Ways to simulate the novel coronavirus: Knowing that both sides need to simulate the novel
coronavirus, both sides summed up a series of pseudovirus packaging methods for both sides
to choose the best packaging method according to their own experimental characteristics.
LZU-CHINA team is concerned about the extensive application of Vesicular stomatitis Virus
(VSV) vector in the study of viral particles invading host cells, identification of cell
surface receptors mediating viral infection, screening of viral inhibitors and vaccine development.
VSV vector can efficiently use the envelope protein of heterologous viruses for presudotypted.
Recombinant VSV is a G glycoprotein gene deletion, which leads to VSV self-replication defects.
G glycoprotein genes are usually replaced by reporter genes for the determination of viral
transduction effect. Considering that the experiments designed by the NMU_China team need to
infect macrophages and determine the subsequent virus clearance effect, and a large number of
timely measurement of transduction effect is needed, the LZU-CHINA team suggested that the
NMU_China team select VSV as the carrier, and replace the G glycoprotein gene with the luciferase
reporter gene, so as to realize the process monitoring of the virus entering the host cells.
At the same time, the summary of the pseudovirus packaging method can also provide reference
for the future iGEM team. Finally, the LZU-CHINA team selected the lentivirus with higher
exogenous gene expression as the vector, and NMU_China selected VSV as the vector.
………………………………………………………………………………… Equation 1
…………………………………………………………………………………………………… Virus concentration
…………………………………………………………………………………………IL-6 concentration function
……………………………………………………………………………………………………Virus growth rate
…………………………………………………………………………………………………Virus attenuation rate
