Microcell-mediated chromosome transfer

Microcell Mediated Chromosome Transfer (or MMCT) is a technique used in cell biology and genetics to transfer a chromosome from a defined donor cell line into a recipient cell line. MMCT has been in use since the 1970s and has contributed to a multitude of discoveries including tumor, metastasis and telomerase suppressor genes as well as information about epigenetics, x-inactivation, mitochondrial function and aneuploidy.[1][2] MMCT follows the basic procedure where donor cells (i.e. cells providing one or more chromosomes or fragments to a recipient cell) are induced to multinucleate their chromosomes. These nuclei are then forced through the cell membrane to create microcells, which can be fused to a recipient cell line.[1]

History

The term MMCT was first used by Fournier and Ruddle in 1977.[3] Their method was based on previous work from 1974 by Ege, Ringertz, Veomett and colleagues,[4][5] synthesizing the techniques used at the time to induce multinucleation in cells, nuclear removal and cell-cell fusions. The next major step in MMCT came during the 1980s when new transfection techniques were utilized to introduce selectable markers onto chromosomes thus making it possible to select for the introduction of specific chromosomes and more easily create defined hybrids.[1]

Procedure

Procedures for MMCT differ slightly but they all require: the induction of multinucleation, enucleation (nuclear removal), and fusion. Multinucleation is usually accomplished through causing prolonged mitotic arrest by colcemid treatment. Certain cells will then "slip" out of mitosis and form multiple nuclei. These nuclei can then be removed using cytochalasin B to disrupt the cytoskeleton and centrifugation in a density gradient to force enucleation. The newly created microcells can then be fused to recipient (target) cells by exposure to poly ethylene glycol, use of Sendai virus, or electrofusion.[1][6]

Variations now allow construction of "humanized" mice with large pieces from human chromosomes[7] as well as new methods for human and mouse artificial chromosomes.[8]

References

  1. 1 2 3 4 Doherty AM, Fisher EM (Sep 2003). "Microcell-mediated chromosome transfer (MMCT): small cells with huge potential". Mamm Genome. 14 (9): 583–92. doi:10.1007/s00335-003-4002-0.
  2. ↑ Sengupta K.; et al. (Feb 2007). "Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells". PLOS ONE. 2 (2): e199. PMC 1805818 Freely accessible. PMID 17332847. doi:10.1371/journal.pone.0000199.
  3. ↑ Fournier RE, Ruddle FH (Jan 1977). "Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells". Proc Natl Acad Sci USA. 74 (1): 319–23. PMC 393251 Freely accessible. PMID 264685. doi:10.1073/pnas.74.1.319.
  4. ↑ Ege T, Ringertz NR (Aug 1974). "Preparation of microcells by enucleation of micronucleate cells". Exp Cell Res. 87 (2): 378–82. PMID 4370277. doi:10.1016/0014-4827(74)90494-7.
  5. ↑ Veomett G, Prescott DM, Shay J, Porter KR (May 1974). "Reconstruction of mammalian cells from nuclear and cytoplasmic components separated by treatment with cytochalasin B". Proc Natl Acad Sci USA. 71 (5): 1999–2002. PMC 388372 Freely accessible. PMID 4525471. doi:10.1073/pnas.71.5.1999.
  6. ↑ Killary AM, Lott ST (Feb 1996). "Production of Microcell Hybrids". Methods. 9 (1): 3–11. PMID 9245337. doi:10.1006/meth.1996.0002.
  7. ↑ Tomizuka K1, Shinohara T, Yoshida H, Uejima H, Ohguma A, Tanaka S, Sato K, Oshimura M, Ishida I (Jan 2000). "Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies". Proc Natl Acad Sci USA. 97 (2): 722–7. PMC 15397 Freely accessible. PMID 10639146. doi:10.1073/pnas.97.2.722.
  8. ↑ Oshimura M, Uno N, Kazuki Y, Katoh M, Inoue T (Feb 2015). "A pathway from chromosome transfer to engineering resulting in human and mouse artificial chromosomes for a variety of applications to bio-medical challenges". Chromosome Res. 23 (1): 111–33. PMC 4365188 Freely accessible. PMID 25657031. doi:10.1007/s10577-014-9459-z.
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