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Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9

  • Authors:
    • Xinyu Ma
    • Ru Cao
    • Haiyan Xiao
    • Zhongwei Cao
  • View Affiliations / Copyright

    Affiliations: Surgical Department of Thyroid Gland, Mammary Gland and Hernia, Inner Mongolia People's Hospital, Hohhot, Inner Mongolia 010010, P.R. China
    Copyright: © Ma et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 354
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    Published online on: October 11, 2020
       https://doi.org/10.3892/ol.2020.12216
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Abstract

Human epidermal growth factor receptor 2 (HER‑2)+ breast cancer has a high recurrence rate and a poor prognosis, with drug resistance contributing to disease progression. The present study aimed to establish a SKBR3 cell line with type II insulin‑like growth factor receptor (IGR‑IIR) gene site integration using the CRISPR/Cas9 system, and to provide a cell model for exploring the mechanism responsible for the effect of IGF‑IIR on trastuzumab resistance in HER‑2+ breast cancer cells. In the present study, six single guide (sg)RNA pairs according to the adeno‑associated virus integration site 1 (AAVS1) gene sequence were designed and synthesized, and the Universal CRISPR Activity assay CRISPR/Cas9 rapid construction and activity detection kit was used to connect the annealed oligo with the pCS vector. The sgRNA with the highest efficiency was selected to construct a Cas9/sgRNA expression vector using AsiSI + Bstz17I restriction enzymes to cut IGF‑IIR. The fragment was ligated into an human AAVS1‑KI vector to construct the IGF‑IIR targeting vector. The Cas9/sgRNA and IGF‑IIR targeting vectors were electroporated into SKBR3 cells, screened using puromycin and identified via PCR, and the mixed cloned cells generated via IGF‑IIR gene targeted integration were obtained. The semi‑solid and limited dilution methods were used for monoclonal cell preparation, and the results revealed that a Cas9/sgRNA vector that targeted the AAVS1 was successfully constructed. sgRNA activity detection demonstrated that sgRNA2 had the highest efficiency, while enzyme digestion and sequencing confirmed that the IGF‑IIR target vector was successfully constructed. The optimum conditions for electrotransfection were 1,200 V, 20 ms and 2 pulses, and the optimal screening concentration of puromycin was 0.5 µg/ml. Using these conditions, the IGF‑IIR targeting vector and pCS‑sgRNA2 plasmid were successfully transfected into SKBR3 cells, and PCR identification and sequencing verified the correct genotype of mixed clone fragments. The monoclonal cells proliferate slowly and gradually underwent apoptosis. Overall, the present study successfully obtained a mixed clone cell line with site‑specific integration of the IGF‑IIR gene at the AAVS1.
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Copy and paste a formatted citation
Spandidos Publications style
Ma X, Cao R, Xiao H and Cao Z: Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9. Oncol Lett 20: 354, 2020.
APA
Ma, X., Cao, R., Xiao, H., & Cao, Z. (2020). Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9. Oncology Letters, 20, 354. https://doi.org/10.3892/ol.2020.12216
MLA
Ma, X., Cao, R., Xiao, H., Cao, Z."Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9". Oncology Letters 20.6 (2020): 354.
Chicago
Ma, X., Cao, R., Xiao, H., Cao, Z."Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9". Oncology Letters 20, no. 6 (2020): 354. https://doi.org/10.3892/ol.2020.12216
Copy and paste a formatted citation
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Spandidos Publications style
Ma X, Cao R, Xiao H and Cao Z: Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9. Oncol Lett 20: 354, 2020.
APA
Ma, X., Cao, R., Xiao, H., & Cao, Z. (2020). Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9. Oncology Letters, 20, 354. https://doi.org/10.3892/ol.2020.12216
MLA
Ma, X., Cao, R., Xiao, H., Cao, Z."Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9". Oncology Letters 20.6 (2020): 354.
Chicago
Ma, X., Cao, R., Xiao, H., Cao, Z."Establishment of a type II insulin‑like growth factor receptor gene site‑integrated SKBR3 cell line using CRISPR/Cas9". Oncology Letters 20, no. 6 (2020): 354. https://doi.org/10.3892/ol.2020.12216
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