Dilution cloning or cloning by limiting dilution [1][2] describes a procedure to obtain a monoclonal cell population starting from a polyclonal mass of cells. This is achieved by setting up a series of increasing dilutions of the parent (polyclonal) cell culture. A suspension of the parent cells is made. Appropriate dilutions are then made, depending on cell number in the starting population, as well as the viability and characteristics of the cells being cloned. After the final dilutions are produced, aliquots of the suspension are plated[1] or placed in wells[2] and incubated. If all works correctly, a monoclonal cell colony will be produced. Applications for the procedure include cloning of parasites,[3] T cells,[4] transgenic cells,[5] macrophages.[6] and hematopoietic stem cells.[7]
References
edit- ^ a b Freshney, R. Ian (2010). Culture of animal cells : a manual of basic technique and specialized applications (6th ed.). Hoboken, N.J.: Wiley-Blackwell. pp. 208–211. ISBN 9780470528129.
- ^ a b Davis, John M, ed. (2011). Animal cell culture. Oxford: Wiley-Blackwell. pp. 239–240. ISBN 978047066658-6.
- ^ Butterworth, Alice S; Robertson, Alan J; Ho, Mei-Fong; Gatton, Michelle L; McCarthy, James S; Trenholme, Katharine R (2011). "An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites". Malaria Journal. 10 (1): 95. doi:10.1186/1475-2875-10-95. ISSN 1475-2875. PMC 3089786. PMID 21496350.
- ^ Rout, Namita; Else, James G.; Yue, Simon; Connole, Michelle; Exley, Mark A.; Kaur, Amitinder (2 July 2010). "Heterogeneity in phenotype and function of CD8+ and CD4/CD8 double-negative Natural Killer T cell subsets in sooty mangabeys". Journal of Medical Primatology. 39 (4): 224–234. doi:10.1111/j.1600-0684.2010.00431.x. PMC 2904642. PMID 20618588.
- ^ Lilienfeld, Benjamin G.; Crew, Mark D.; Forte, Pietro; Baumann, Bettina C.; Seebach, Jörg D. (1 March 2007). "Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity". Xenotransplantation. 14 (2): 126–134. doi:10.1111/j.1399-3089.2007.00378.x. PMID 17381687. S2CID 1433274.
- ^ Seki, Yoshihiro; Suzuki, Satoshi O.; Masui, Kenta; Harada, Shiori; Nakamura, Seiji; Kanba, Shigenobu; Iwaki, Toru (1 June 2011). "A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film". Neuropathology. 31 (3): 215–222. doi:10.1111/j.1440-1789.2010.01163.x. PMID 21092060. S2CID 26016732.
- ^ Sieburg, Hans B; Cho, Rebecca H; Muller-Sieburg, Christa E (2002). "Limiting dilution analysis for estimating the frequency of hematopoietic stem cells: uncertainty and significance". Exp Hematol. 39 (12): 1436–1443. doi:10.1016/s0301-472x(02)00963-3 – via ISEH.
External links
edit- Robert Staszewski "Cloning by Limiting Dilution: an improved estimate that an interesting culture is monoclonal" [1]
- John A. Ryan "Cell Cloning by Serial Dilution in 96 Well Plates".
- Vilma Maldonado,Jorge Meléndez-Zajgla "A modified method for cloning adherent mammalian cells".
- "Cloning by Limiting Dilution".[permanent dead link]
- "Cloning by limiting dilution".
- Nanci Donacki "Cloning by Limiting Dilution of Hybridoma".