Stable silencing of IGF1R using Lentiviral-mediated shRNA in HEK293T cells
L Nasehi1, M H Ghahremani1, K Yavari2
1Department of Molecular Medicine, School of Advance Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Abstract:
Insulin-like growth factors are among the peptide mitogens that regulate cell proliferation and differentiation as well as mediator of antiapoptotic signals. The imbalance between the expression and activities of these molecules may lead to malignancy in cells. Evidences have suggested the insulin-like growth factor 1 receptor (IGF-1R) signaling pathway as a therapeutic target in the management and treatment of cancer. In this present study, we have generated silencing stable clones of HEK cells using six different pGIPZ (lentiviral vector) shRNAs targeted to human IGF-1R gene and a pGIPZ non-silencing shRNAmir lentiviral vector (as negative control). The recombinant lentiviral vectors were separately transduced into human embryonic kidney 293 T (HEK293T) cell lines. The knockdown of IGF-1R was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and the relative IGF-1R mRNA levels were expressed as a ratio of IGF-1R to β-actin by REST software. The results showed significant reduction in the expression of IGF-1R mRNAs in cells transduced with all six pGIPZ-IGF-1R recombinant lentivirals compared to non-silencing negative control. No significant difference was observed among the six cassettes. Results indicated that recombinant lentiviral vectors provided an efficient and stable knockdown of IGF-1R providing useful tool for IGF-1R pathway studies.
Insights
This study demonstrates that lentiviral vectors effectively silence the insulin-like growth factor 1 receptor (IGF-1R) in HEK293T cells. This provides a valuable tool for investigating the IGF-1R pathway in cancer research.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Insulin-like growth factors (IGFs) regulate cell growth and survival.
- Dysregulation of IGF signaling is implicated in cancer development.
- The IGF-1 receptor (IGF-1R) pathway is a potential therapeutic target for cancer treatment.
Purpose of the Study:
- To develop and validate a method for stable knockdown of the IGF-1 receptor (IGF-1R) in human embryonic kidney (HEK293T) cells.
- To assess the efficacy of lentiviral short hairpin RNAs (shRNAs) in reducing IGF-1R expression.
Main Methods:
- Generation of stable HEK293T cell clones using six different pGIPZ lentiviral shRNAs targeting the human IGF-1R gene.
- Transduction of HEK293T cells with recombinant lentiviral vectors.
- Confirmation of IGF-1R knockdown using reverse transcription polymerase chain reaction (RT-PCR).
- Quantification of relative IGF-1R mRNA levels using REST software.
Main Results:
- Significant reduction in IGF-1R mRNA expression was observed in cells treated with all six pGIPZ-IGF-1R lentivirals compared to the non-silencing control.
- No significant differences in knockdown efficiency were noted among the six shRNA constructs.
- Lentiviral vectors demonstrated efficient and stable knockdown of IGF-1R.
Conclusions:
- Lentiviral vector-mediated shRNA delivery is an effective strategy for achieving stable IGF-1R knockdown in HEK293T cells.
- This approach provides a robust tool for further research into the role of the IGF-1R pathway in cellular processes and disease.
- The developed system facilitates studies on IGF-1R function and its potential as a therapeutic target.
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