|
| Status |
Public on Sep 30, 2024 |
| Title |
Escaping from a dominant‐negative KRAS using REPLACE |
| Organism |
Mesocricetus auratus |
| Experiment type |
Other
|
| Summary |
A dominant-negative gene therapy approach has been proposed and tested on proto-oncogene KRAS, wherein the oncogenic activity (and cell proliferation) of KRAS can be suppressed by introducing a dominant-negative KRAS allele (S17N). We employed REPLACE to conduct continuous evolution on KRAS (S17N) and examined its potential pathways for conferring resistance in this gene therapy methodology.
|
| |
|
| Overall design |
KRAS (S17N) cDNA was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and the resulting RNAs were electroporated into aforementioned host cells. Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. The remaining cells were partially cryopreserved as backup and partially utilized for RNA extraction and mutation analysis. After another 9 d of culture, cells were harvested for total RNA extraction, sequencing, and mutation analysis. As a control, analogous experiments were conducted utilizing wild-type KRAS.
|
| |
|
| Contributor(s) |
Lin Y, Ma L |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| |
| Submission date |
Jul 09, 2024 |
| Last update date |
Sep 30, 2024 |
| Contact name |
Yihan Lin |
| E-mail(s) |
yihan.lin@pku.edu.cn
|
| Organization name |
Peking University
|
| Street address |
No.5 Yiheyuan Road, Haidian
|
| City |
Beijing |
| ZIP/Postal code |
10087 |
| Country |
China |
| |
|
| Platforms (1) |
| GPL29575 |
Illumina MiSeq (Mesocricetus auratus) |
|
| Samples (12)
|
|
| This SubSeries is part of SuperSeries: |
| GSE235343 |
Replicative RNA enables directed evolution and Darwinian adaptation in mammalian cells |
|
| Relations |
| BioProject |
PRJNA1133794 |
Less...
More...