Skip to main content
SpringerLink
Log in

The L-Cell Isolation from Heterogonous Population of Intestinal Cell Line Using Antibiotic Selection Method

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Enteroendocrine cells are the largest population of hormone-producing cells in the body and play important roles in many aspects of body functions. The enteroendocrine cell population is divided into different subpopulations that secrete different hormones and peptides. Characterization of each subpopulation is particularly useful for analyzing the cellular mechanisms responsible for specific cell types. Therefore, the necessity of a pure cell line for a specific study purpose was the important motivation for the separation of cell lines for each subpopulation of enteroendocrine cells. The present research introduces a method for the isolation of L-cells, one of the important subpopulations of enteroendocrine cells. The antibiotic selection method was conducted in order to isolate the L-cells from a heterogonous population of intestinal cell line. In this method, a neomycin resistance gene (as selected marker) was expressed under the control of a specific promoter of L-cells. After transfection of manipulated plasmid, only the cells which determine the specific promoter and express neomycin resistance protein would be able to survive under Geneticin antibiotic treatment condition. In order to confirm that the isolated cells were L-cells, reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR assays were performed. Based on the results, the isolated cells were pure L-cells that could be able to express specific mRNA of L-cells efficiently. This technique provides a unique method for the isolation and purification of any cell line. The purified isolated L-cells by this method can be used for future studies and for analyzing cellular mechanisms that involve L-cells' functions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Stachura, J., Krause, W. J., & Ivey, K. J. (1981). Ultrastructure of endocrine-like cells in lamina propria of human gastric mucosa. Gut, 22, 534–541.

    Article  CAS  Google Scholar 

  2. Theodorakis, M. J., Carlson, O., Michopoulos, S., Doyle, M. E., Juhaszova, M., Petraki, K., & Egan, J. M. (2006). Human duodenal enteroendocrine cells: source of both incretin peptides, GLP-1 and GIP. American journal of Physiology - Endocrinology and Metabolism , 290, E550–E559.

    Article  CAS  Google Scholar 

  3. Girard, J. (2008). The incretins: from the concept to their use in the treatment of type 2 diabetes. Part A: incretins: concept and physiological functions. Diabetes & metabolism, 34, 550–559.

    Article  CAS  Google Scholar 

  4. Schonhoff, S. E., Giel-Moloney, M., & Leiter, A. B. (2004). Minireview: development and differentiation of gut endocrine cells. Endocrinology, 145, 2639–2644.

    Article  CAS  Google Scholar 

  5. Martin, G. R., Beck, P. L., & Sigalet, D. L. (2006). Gut hormones, and short bowel syndrome: the enigmatic role of glucagon-like peptide-2 in the regulation of intestinal adaptation. World Journal of Gastroenterology, 12, 4117–4129.

    CAS  Google Scholar 

  6. McDermott, J. R., Leslie, F. C., D'Amato, M., Thompson, D. G., Grencis, R. K., & McLaughlin, J. T. (2006). Immune control of food intake: enteroendocrine cells are regulated by CD4+ T lymphocytes during small intestinal inflammation. Gut, 55, 492–497.

    Article  CAS  Google Scholar 

  7. Moran, G. W., Leslie, F. C., Levison, S. E., Worthington, J., & McLaughlin, J. T. (2008). Enteroendocrine cells: neglected players in gastrointestinal disorders? Therapeutic Advances in Gastroenterology, 1, 51–60.

    Article  Google Scholar 

  8. Burcelin, R. (2005). The incretins: a link between nutrients and well-being. The British Journal of Nutrition, 93(Suppl 1), S147–S156.

    Article  CAS  Google Scholar 

  9. During, M. J., Cao, L., Zuzga, D. S., Francis, J. S., Fitzsimons, H. L., Jiao, X., Bland, R. J., Klugmann, M., Banks, W. A., Drucker, D. J., et al. (2003). Glucagon-like peptide-1 receptor is involved in learning and neuroprotection. Nature Medicine, 9, 1173–1179.

    Article  CAS  Google Scholar 

  10. Mattson, M. P., Perry, T., & Greig, N. H. (2003). Learning from the gut. Nature Medicine, 9, 1113–1115.

    Article  CAS  Google Scholar 

  11. Rasouli, M., Ahmad, Z., Omar, A. R., & Allaudin, Z. N. (2011). Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment. BMC Biotechnology, 11, 99.

    Article  CAS  Google Scholar 

  12. Cheung, A. T., Dayanandan, B., Lewis, J. T., Korbutt, G. S., Rajotte, R. V., Bryer-Ash, M., Boylan, M. O., Wolfe, M. M., & Kieffer, T. J. (2000). Glucose-dependent insulin release from genetically engineered K cells. Science, 290, 1959–1962.

    Article  CAS  Google Scholar 

  13. Vilsboll, T., Krarup, T., Madsbad, S., & Holst, J. J. (2003). Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects. Regulatory Peptides, 114, 115–121.

    Article  CAS  Google Scholar 

  14. Seidah, N. G., Chretien, M., & Day, R. (1994). The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Biochimie, 76, 197–209.

    Article  CAS  Google Scholar 

  15. Lynch, D. R., Strittmatter, S. M., Venable, J. C., & Snyder, S. H. (1987). Enkephalin convertase in the gastrointestinal tract an associated organs characterized and localized with [3H]guanidinoethylmercaptosuccinic acid. Endocrinology, 121, 116–126.

    Article  CAS  Google Scholar 

  16. Ramshur, E. B., Rull, T. R., & Wice, B. M. (2002). Novel insulin/GIP co-producing cell lines provide unexpected insights into gut K-cell function in vivo. Journal of Cellular Physiology, 192, 339–350.

    Article  CAS  Google Scholar 

  17. Rindi, G., Grant, S. G., Yiangou, Y., Ghatei, M. A., Bloom, S. R., Bautch, V. L., Solcia, E., & Polak, J. M. (1990). Development of neuroendocrine tumors in the gastrointestinal tract of transgenic mice. Heterogeneity of hormone expression. The American Journal of Pathology, 136, 1349–1363.

    CAS  Google Scholar 

  18. Wice, B. M., & Gordon, J. I. (1995). A tetraspan membrane glycoprotein produced in the human intestinal epithelium and liver that can regulate cell density-dependent proliferation. The Journal of Biological Chemistry, 270, 21907–21918.

    Article  CAS  Google Scholar 

  19. Drucker, D. J., Jin, T., Asa, S. L., Young, T. A., & Brubaker, P. L. (1994). Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. Molecular Endocrinology, 8, 1646–1655.

    CAS  Google Scholar 

  20. Gosmain, Y., Avril, I., Mamin, A., & Philippe, J. (2007). Pax-6 and c-Maf functionally interact with the alpha-cell-specific DNA element G1 in vivo to promote glucagon gene expression. The Journal of Biological Chemistry, 282, 35024–35034.

    Article  CAS  Google Scholar 

  21. Amos, P. J., Cagavi Bozkulak, E., & Qyang, Y. (2012). Methods of cell purification: a critical juncture for laboratory research and translational science. Cells, Tissues, Organs, 195, 26–40.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This project was supported by the E-Science Fund from the Ministry of Science, Technology and Innovation, Malaysia under award number 02-01-04-SF1056. We would like to thank Prof. Douglas Hanahan from the Department of Biochemistry and Biophysics, University of California San Francisco, USA for providing us the STC-1 cell line and also Dr. Yvan Gosmain from the Diabetes Unit, University Hospital, University of Geneva Medical School, Geneva, Switzerland for providing us the Glu.BS plasmid. We also would like to thank Dr. Tan Sheau Wei from the Laboratory of Vaccines and Immunotherapeutics, Universiti Putra Malaysia for assisting us in the real-time PCR work.

Author information

Authors and Affiliations

  1. Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, 43400, Serdang, Selangor, Malaysia

    Mina Rasouli & Abdul Rahman Omar

  2. Department of Biotechnology, School of Allied Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Sakineh Abbasi

  3. Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia

    Kazhal Sarsaifi

  4. Molecular Biomedicine Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia

    Homayoun Hani

  5. Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia

    Zalinah Ahmad

Authors
  1. Mina Rasouli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sakineh Abbasi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kazhal Sarsaifi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Homayoun Hani
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zalinah Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Abdul Rahman Omar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mina Rasouli.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rasouli, M., Abbasi, S., Sarsaifi, K. et al. The L-Cell Isolation from Heterogonous Population of Intestinal Cell Line Using Antibiotic Selection Method. Appl Biochem Biotechnol 172, 394–404 (2014). https://doi.org/10.1007/s12010-013-0514-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-013-0514-6

Keywords

Search

Navigation