Limiting dilution cloning

The dilution cloning (to German limiting dilution cloning ) is a method for isolating a clone . A monoclonal cell culture can be started with the clone .

principle

Limiting Dilution Cloning is based on the dilution of a suspension of cells in several dilution levels up to limiting dilution , in which statistically only one or no cells are present in a dilution level. As a result of the isolated cultivation of this single cell, all daughter cells that arise from it are genetically identical clones. In the case of animal cells, it is often necessary to use feeding cells at the same time . Tissue cells have, as adherent cells, cell-cell contacts which, before isolation, B. be solved with trypsin .

Cloning based on limiting dilution is used to isolate cells, e.g. B. in hybridomas , in transgenic organisms after cloning , in induced pluripotent stem cells , in parasites , in T cells , or in macrophages .

Alternatives to isolation based on limiting dilution are e.g. B. FACS , MACS or manual isolation with microcapillaries under an inverted microscope.

literature

Friedrich Lottspeich , Joachim W. Engels , Solodkoff Zettlmeier Lay: Bioanalytik . 3rd edition, Spektrum, Heidelberg, 2012, ISBN 978-3-8274-0041-3 .
Cornel Mülhardt: The Experimenter: Molecular Biology / Genomics. Sixth edition. Spectrum Akademischer Verlag, Heidelberg 2008, ISBN 3-8274-2036-9 .
J. Sambrook , T. Maniatis , DW Russel: Molecular cloning: a laboratory manual. 3rd edition (2001), Cold Spring Harbor Laboratory Press. ISBN 0-87969-577-3 .
R. Staszewski: Cloning by limiting dilution: an improved estimate that an interesting culture is monoclonal. In: The Yale journal of biology and medicine. Volume 57, Number 6, 1984 Nov-Dec, pp 865-868, PMID 6537695 , PMC 2589791 (free full text).

Web links

John A. Ryan: Cell Cloning by Serial Dilution in 96 Well Plates. (PDF file)
Vilma Maldonado, Jorge Meléndez-Zajgla: A modified method for cloning adherent mammalian cells.
Cloning by limiting dilution.
Nanci Donacki Cloning by Limiting Dilution of Hybridoma.

Individual evidence

↑ R. Ian Freshney: Culture of animal cells: a manual of basic technique and specialized applications , 6th ed .. Edition, Wiley-Blackwell, Hoboken, NJ 2010, ISBN 978-0-470-52812-9 , pp 208- 211.
↑ edited by John M Davis: Animal cell culture . Wiley-Blackwell, Oxford 2011, ISBN 978-0-470-66658-6 , pp. 239-240.
^ Benjamin G. Lilienfeld, Crew, Mark D., Forte, Pietro, Baumann, Bettina C., Seebach, Jörg D .: Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity . In: Xenotransplantation . 14, No. 2, March 1, 2007, pp. 126-134. doi : 10.1111 / j.1399-3089.2007.00378.x .
↑ Alice S Butterworth, Robertson, Alan J, Ho, Mei-Fong, Gatton, Michelle L, McCarthy, James S, Trenholme, Katharine R: An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites . In: Malaria Journal . 10, No. 1, January 1, 2011, p. 95. doi : 10.1186 / 1475-2875-10-95 .
↑ Namita Rout, Else, James G., Yue, Simon, Connole, Michelle, Exley, Mark A., Kaur, Amitinder: Heterogeneity in phenotype and function of CD8 + and CD4 / CD8 double-negative Natural Killer T cell subsets in sooty mgenauys . In: Journal of Medical Primatology . 39, No. 4, July 2, 2010, pp. 224-234. doi : 10.1111 / j.1600-0684.2010.00431.x .
↑ Yoshihiro Seki, Suzuki, Satoshi O., Masui, Kenta, Harada, Shiori, Nakamura, Seiji, Kanba, Shigenobu, Iwaki, Toru: A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film . In: Neuropathology . 31, No. 3, June 1, 2011, pp. 215-222. doi : 10.1111 / j.1440-1789.2010.01163.x .

[Fresh-1] R. Ian Freshney: Culture of animal cells: a manual of basic technique and specialized applications , 6th ed .. Edition, Wiley-Blackwell, Hoboken, NJ 2010, ISBN 978-0-470-52812-9 , pp 208- 211.

[Dav-2] ted by John M Davis: Animal cell culture . Wiley-Blackwell, Oxford 2011, ISBN 978-0-470-66658-6 , pp. 239-240.

[3] Benjamin G. Lilienfeld, Crew, Mark D., Forte, Pietro, Baumann, Bettina C., Seebach, Jörg D .: Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity . In: Xenotransplantation . 14, No. 2, March 1, 2007, pp. 126-134. doi : 10.1111 / j.1399-3089.2007.00378.x .

[4] Alice S Butterworth, Robertson, Alan J, Ho, Mei-Fong, Gatton, Michelle L, McCarthy, James S, Trenholme, Katharine R: An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites . In: Malaria Journal . 10, No. 1, January 1, 2011, p. 95. doi : 10.1186 / 1475-2875-10-95 .

[5] Namita Rout, Else, James G., Yue, Simon, Connole, Michelle, Exley, Mark A., Kaur, Amitinder: Heterogeneity in phenotype and function of CD8 + and CD4 / CD8 double-negative Natural Killer T cell subsets in sooty mgenauys . In: Journal of Medical Primatology . 39, No. 4, July 2, 2010, pp. 224-234. doi : 10.1111 / j.1600-0684.2010.00431.x .

[6] Yoshihiro Seki, Suzuki, Satoshi O., Masui, Kenta, Harada, Shiori, Nakamura, Seiji, Kanba, Shigenobu, Iwaki, Toru: A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film . In: Neuropathology . 31, No. 3, June 1, 2011, pp. 215-222. doi : 10.1111 / j.1440-1789.2010.01163.x .