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Updated: Aug 27, 2025
Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation
Published on: February 28, 2025
Construction of TSC2 knockout cell line using CRISPR/Cas9 system and demonstration of its effects on NIH-3T3 cells
Xu Wang1, Yang Zhao1, Zhan Wang1
1Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China.
Abstract:
Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder involving multiple organ systems. TSC2 gene plays an important role in the development of TSC. The most common kidney manifestation of TSC is renal angiomyolipoma (RAML). TSC-RAML is more likely to be bilateral multiple tumors and tends to destroy the renal structure and damages renal function severely. As a result, patients with TSC-RAML often miss the opportunity for surgical treatment when TSC-RAML is diagnosed, causing difficulty in obtaining tumor specimens through surgery. Due to this difficulty, model cell lines must be constructed for scientific research. In this paper, TSC2 was knocked out in NIH-3T3 cell lines by CRISPR/Cas9 system. PCR, WB and mTOR inhibitor drug sensitivity test showed that the TSC2 knockout NIH-3T3 cells were successfully constructed. The ability of proliferation and invasion in TSC2 KO NIH-3T3 cells were higher than those in wild type group. The constructed KO cell line lay the foundation for further study of TSC.
Insights
Researchers created a new cell model for Tuberous Sclerosis Complex (TSC) by knocking out the TSC2 gene in NIH-3T3 cells. This model will aid further research into TSC and its kidney manifestations, like renal angiomyolipoma.
Area of Science:
- Genetics
- Oncology
- Cell Biology
Background:
- Tuberous Sclerosis Complex (TSC) is a rare genetic disorder affecting multiple organs.
- Renal angiomyolipoma (RAML) is a common and severe kidney complication of TSC.
- Difficulty in obtaining surgical specimens hinders TSC research.
Purpose of the Study:
- To establish a TSC2-knockout NIH-3T3 cell line for studying TSC.
- To provide a reliable model for investigating TSC pathogenesis and potential therapies.
Main Methods:
- CRISPR/Cas9 gene editing was used to knock out the TSC2 gene in NIH-3T3 cells.
- Polymerase Chain Reaction (PCR) and Western Blot (WB) confirmed gene knockout.
- mTOR inhibitor drug sensitivity assays were performed.
Main Results:
- Successful construction of TSC2-knockout NIH-3T3 cell line confirmed by PCR and WB.
- TSC2 knockout cells exhibited increased proliferation and invasion compared to wild-type cells.
- The new cell line showed altered sensitivity to mTOR inhibitors.
Conclusions:
- The developed TSC2-knockout cell line serves as a valuable tool for TSC research.
- This model facilitates the study of TSC-associated cellular behaviors, including proliferation and invasion.
- Further investigations into TSC pathogenesis and therapeutic strategies can be advanced using this cell line.
