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Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
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Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

DOI:
10.3791/56726-v

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10:21 min

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January 05, 2018

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,
1Division of Hematology, Department of Medicine,Brigham and Women’s Hospital, Harvard Medical School, 2Broad Institute, 3Dana-Farber Cancer Institute,Harvard Medical School

Chapters

  • 00:05Title
  • 00:44Cloning sgRNA Oligos
  • 01:08Digestion of LentiGuide-puroVector
  • 02:04Ligation of Annealed Oligos into Digested Backbone
  • 03:02Generation of Cell Lines Stably Expressing spCas9
  • 05:40Reporter Assay for Cas9 Activity
  • 06:10Spinfection of sgRNAs into Cas9 Expressing Cells
  • 06:31Ba/F3 Cellular Transformation and Positive Selection using m-IL3 Withdrawal
  • 07:23Results: Use of CRISPR/Cas9 Tool and Ba/F3 Cellular Transformation to Study Oncogenic Potential of Mutated Calreticulin
  • 09:02Conclusion

Summary

자동 번역

자동 번역

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Tags

Keywords: CRISPR/Cas9Gene EditingCalreticulinOncogenic ActivityCytokine Dependent Hematopoietic CellsHematological MalignanciesLentiGuide-Puro VectorBsmB1 Restriction EnzymePhosphataseLigationSTBL3 CellsHEK293T CellsTransfection

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