Dilution cloning

Dilution cloning (also called cloning by limiting dilution or simply limiting dilution) is a technique used to derive a monoclonal population from a heterogeneous cell mixture by distributing cells at sufficiently low density that some culture compartments (e.g., wells) contain a single viable cell. After expansion, wells seeded by one cell yield clonal progeny. The approach is widely used for isolating hybridomas and other mammalian cell clones, and has applications in immunology, microbiology, parasitology and stem cell biology.[1][2]

Principle

Limiting dilution relies on the Poisson distribution to set cell input so that a significant fraction of culture wells receive exactly one cell (and many receive none). After culture under suitable conditions (often with feeder cells and growth factors), wells are screened to identify monoclonal outgrowths. Statistical frameworks for dose–response design, analysis and estimation of clone-initiating cell frequencies are detailed in the immunology literature.[3]

History

Early forms of limiting dilution for obtaining pure microbial cultures date to the 19th century, and cloning of bacteria by isolation of single colonies on solid media was established by the late 1800s.[4]

In immunology, the modern limiting-dilution microculture system for cloning and quantifying rare B- and T-lymphocyte precursors was developed in the 1970s in the laboratory of Ivan Lefkovits at the Basel Institute for Immunology and was subsequently formalized with Herman Waldmann; their monograph remains a primary reference for the method.[3][5][6] Reviews in the 1980s outlined the theoretical basis and experimental implementations for lymphocyte cloning and frequency analysis.[7][8] Lefkovits is widely credited with developing the microculture technique for inducing antibody-forming cells in limiting dilution cultures of mouse spleen cells.[9]

Applications

Dilution cloning is used to:

  • derive monoclonal hybridoma and recombinant cell lines for antibody production;[1]
  • clone T cells and B cells for functional studies and epitope specificity mapping (often with feeder cells and cytokines);[3][10]
  • isolate parasites and other microbes at the single-organism level;[11]
  • estimate frequencies of rare precursor cells (e.g., cytotoxic T-lymphocyte or hematopoietic stem cell precursors) using statistical LDA frameworks.[12][13]

See also

References

  1. ^ a b Freshney, R. Ian (2015). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (7th ed.). Wiley-Blackwell. ISBN 978-1-118-87634-6. {{cite book}}: Check |isbn= value: checksum (help)
  2. ^ 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". Experimental Hematology. 30 (12): 1436–1443. doi:10.1016/s0301-472x(02)00968-2. PMID 12482502.
  3. ^ a b c Lefkovits, Ivan; Waldmann, Herman (1999). Limiting Dilution Analysis of Cells of the Immune System (2nd ed.). Oxford University Press. ISBN 0-19-850128-5.
  4. ^ Weiss, Robin A. (2005). "Robert Koch: the grandfather of cloning?". Cell. 123 (5): 735–738. doi:10.1016/j.cell.2005.11.006. PMID 16325570.
  5. ^ Benner, R. (2005). "Dissection of systems, cell populations and molecules". Scandinavian Journal of Immunology. 61 (4): 299–303. doi:10.1111/j.1365-3083.2005.01611.x. PMID 15953186.
  6. ^ Luzzati, Anna L. (2005). "Origin and development of a scientific collaboration". International Archives of Allergy and Immunology. 138 (2): 129–133. doi:10.1159/000086183. PMID 15953179.
  7. ^ Lefkovits, Ivan; Waldmann, Herman (1984). "Limiting dilution analysis of the cells of immune system I: the clonal basis of the immune response". Immunology Today. 5 (9): 265–268. doi:10.1016/0167-5699(84)90137-3. PMID 25290326.
  8. ^ Waldmann, Herman (1984). "Limiting dilution analysis of cells of the immune system II". Immunology Today. 5 (10): 298–303. doi:10.1016/0167-5699(84)90154-3. PMID 25290547.
  9. ^ "Pictures from the people and sciences of the Basel Institute for Immunology". What is Biotechnology?. The Biomedical & Life Sciences Innovation Board. Retrieved 25 October 2025.
  10. ^ Waldmann, H.; Pope, H.; Lefkovits, I. (1975). "Limiting dilution analysis of helper T-cell function". Scandinavian Journal of Immunology. 4 (1): 135–144. doi:10.1111/j.1365-3083.1975.tb02675.x. PMC 1445909. PMID 50413.
  11. ^ Butterworth, Alice S.; Robertson, Alan J.; Ho, Mei-Fong; Gatton, Michelle L.; McCarthy, James S. (2011). "An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites". Malaria Journal. 10 (2) 95. doi:10.1186/1475-2875-10-95. PMC 3078993. PMID 21457528.
  12. ^ Kaminski, Edward; Hows, Jill; Marti, G. E. (1991). "Optimising a limiting dilution culture system for quantitating frequencies of alloreactive cytotoxic T lymphocyte precursors". Cellular Immunology. 134 (1): 210–222. doi:10.1016/0008-8749(91)90059-K. PMID 1681904.
  13. ^ 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". Experimental Hematology. 30 (12): 1436–1443. doi:10.1016/s0301-472x(02)00968-2. PMID 12482502.
This article is issued from Wikipedia. The text is available under Creative Commons Attribution-Share Alike 4.0 unless otherwise noted. Additional terms may apply for the media files.