Obtaining a pancreatic cancer cell line stably expressing doxycycline-dependent endonuclease Cas9

A.K. Nurgalieva^*, V.S. Skripova^**, L.F. Minigulova^***, R.G. Kiyamova^****

Kazan Federal University, Kazan, 420008 Russia

E-mail: ^*alsina97@mail.ru, ^**vsk190@gmail.com, ^***minigulovalf@gmail.com, ^****kiyamova@mail.ru

Received November 29, 2017

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Abstract

Pancreatic cancer is one of the most painful and aggressive types of cancer with high lethality rate. Early diagnosis of the disease is limited and antitumor therapy has low efficacy, because pancreatic cancer shows multiple drug resistance. It is still unclear how pancreatic tumor cells become resistant to chemotherapy and understanding of the resistance mechanisms and identification of their regulating genes is necessary to develop a new and more efficient pancreatic cancer therapy. One of the methods to identify such genes is CRISPR/Cas9 technology (clustered regularly interspaced short palindromic repeats) where Cas9 endonuclease is used to make double-strand DNA breaks in target genes.

The aim of this work is modification of the pancreatic cancer cell line MIA PaCa-2 to express a doxycycline-inducible Cas9 endonuclease gene. Several plasmid vectors have been used to create a lentivirus containing the Cas9 endonuclease gene. The virus has been used for transduction of tumor cells to get doxycycline-inducible Cas9 expressing clones. Cas9 endonuclease expression has been detected by the western blot analysis with antibodies to FLAG epitope.

Two tumor clones of the MIA Pa-Ca2/Cas9 cell line expressing Cas9 endonuclease have been generated. These cells can be used further to search genes related to regulation of the susceptibility to antitumor drugs.

Keywords: pancreatic cancer, CRISPR/Cas9, Cas9 endonuclease, MIA PaCA-2

Acknowledgments. The study was supported by the Russian Science Foundation (project no. 15-15-20032) and performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

Figure Captions

Fig. 1. The scheme of work on obtaining the pancreatic cancer cell line MIA PaCa-2 / Cas9.

Fig. 2. The western blot analysis of the soluble protein fractions of lysates from the MIA PaCa-2/Cas9 cell line: 1 – MagicMark XP Western Protein Standard (LC5602; Invitrogen, USA); 2, 4 – induction with doxycycline 1 mkg/ml; 3, 5 – non-induced control.

Fig. 3. The western blot analysis of the soluble protein fractions of lysates from the individual clones of the MIA PaCa-2/Cas9 cell line: 1, 8 – MagicMark XP Western Protein Standard (LC5602; Invitrogen, USA); soluble cell lysates: 2 – clone D8 “–” doxycycline; 3 – Clone D8 “+” doxycycline; 4 – clone G10 “–” doxycycline; 5 – clone G10 “+” doxycycline; 6 – general cell population of MIA PaCa-2/ Cas9 “–” doxycycline; 7 – general cell population of MIA PaCa-2 / Cas9 “+” doxycycline; 9 – clone B7 “–” doxycycline; 10 – clone B7 “+” doxycycline; 11 – clone B8 “–” doxycycline; 12 – clone B8 “+” doxycycline; 13 – clone C7 “–” doxycycline; 14 – clone C7 “+” doxycycline.

References

1. Baines A.T., Martin P.M., Rorie C.J. Current and emerging targeting strategies for treatment of pancreatic cancer. Prog. Mol. Biol. Transl. Sci. 2016, vol. 144, pp. 277–320. doi: 10.1016/bs.pmbts.2016.09.006.

2. Liu H., Li L., Chen H., Kong R., Pan S., Hu J., Wang Y., Li Y., Sun B. Silencing IGFBP-2 decreases pancreatic cancer metastasis and enhances chemotherapeutic sensitivity. Oncotarget, 2017, vol. 8, no. 37, pp. –. doi: 10.18632/oncotarget.18669.

3. Li J., Wientjes M.G., Au J.L. Pancreatic cancer: Pathobiology, treatment options, and drug delivery. AAPS J., 2010, vol. 12, no. 2, pp. 223–232. doi: 10.1208/s12248-010-9181-5.

4. Smirnov A.V., Yunusova A.M., Lukyanchikova V.A., Battulin N.R. CRISPR/Cas9, a universal tool for genomic engineering. Russ. J. Genet.: Appl. Res., 2017, vol. 7, no. 4, pp. 440–458.10.1134/S2079059717040116.

5. Bi H., Yang B. Gene editing with TALEN and CRISPR/Cas in rice. Prog. Mol. Biol. Transl. Sci.149, pp. 81–98. doi: 10.1016/bs.pmbts.2017.04.006.

6. Grober N.A., Sukhan T.O., Kuznetsova E.I. CRISPR/CAS9 – a system of genome editing using nucleofection technology. Biotekhnologiya: sostoyanie i perspektivy razvitiya: Materialy IX Mezhdunar. kongressa [Biotechnology: State and Prospects of Development: Proc. IX Int. Conf.], 2017, vol. 1, pp. 265–266.

7. Skripova V., Serebriiskii I., Abramova Z., Astsaturov I., Kiyamova R. CRISPR/Cas9 technique for 2016, vol. 7, no. 1, pp. 97100.

8. Gaponova A.V., Deneka A.Y., Beck T.N., Liu H., Andrianov G., Nikonova A.S., Golemis E.A., Serebriiskii I.G. Identification of evolutionarily conserved DNA damage response genes that al­ter sensitivity to cisplatin. Oncotarget, 2017, vol. 8, no. 12, pp.–

9. Wang T., Wei J.J., Sabatini D.M., Lander E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science, 2014, vol. 343, pp. 8084.

10. Ryan J.A. Cell Cloning by Serial Dilution 96 Well Plates Protocol. Life Sciences, 2008. Available at: http://csmedia2.corning.com/LifeSciences/media/pdf/Single_cell_cloning_protocol.pdf.

For citation: Nurgalieva A.K., Skripova V.S., Minigulova L.F., Kiyamova R.G. Obtaining a pancreatic cancer cell line stably expressing doxycycline-dependent endonuclease Cas9. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2018, vol. 160, no. 3, pp. 386–394. (In Russian)

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