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利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

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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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利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

DOI:

10.3791/56726-v

•

10:21 min

•

January 05, 2018

•

Nouran S. Abdelfattah, Ann Mullally^2,3

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

Capítulos

00:05Título
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

Tadução automática

有针对性的基因编辑使用 CRISPR/Cas9 极大地促进了对基因的生物功能的理解。在这里, 我们利用 CRISPR/Cas9 的方法, 模型钙突变的细胞因子依赖性造血细胞, 以研究其致癌活动。

Tags

Keywords: CRISPR/Cas9 Gene Editing Calreticulin Oncogenic Activity Cytokine Dependent Hematopoietic Cells Hematological Malignancies LentiGuide-Puro Vector BsmB1 Restriction Enzyme Phosphatase Ligation STBL3 Cells HEK293T Cells Transfection

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