The transfer of human artificial chromosomes via cryopreserved microcells
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Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at −80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.
KeywordsChromosome Human artificial chromosome Microcell-mediated chromosome transfer HAC Cancer Synthetic biology Gene delivery
Human artificial chromosome
P1 phage-derived artificial chromosome
Bacterial artificial chromosome
Yeast artificial chromosome
Induced pluripotent stem cell
Mesenchemal stem cell
Microcell mediated chromosome transfer
Microcell mediated chromosome transfer using Measles virus fusogen
Enhanced green fluorescent protein
This study was supported by Regional Innovation Strategy Support Program from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (M. O.), Japan Science and Technology Agency, CREST (M. O.).
N. U. designed and performed most of the experiments. K. U., S. Z., K. U. and H. M. performed the experiments and analyzed the data. M. O. designed the experiments and supervised the entire project. N. U. and M. O. wrote the manuscript.
Conflict of interest
The authors declare no conflicts of interest.
- Hiratsuka M, Uno N, Ueda K, Kurosaki H, Imaoka N, Kazuki K, Ueno E, Akakura Y, Katoh M, Osaki M, Kazuki Y, Nakagawa M, Yamanaka S, Oshimura M (2011) Integration-free iPS cells engineered using human artificial chromosome vectors. PLoS ONE 6:e25961. doi: 10.1371/journal.pone.0025961 CrossRefGoogle Scholar
- Hoshiya H, Kazuki Y, Abe S, Takiguchi M, Kajitani N, Watanabe Y, Yoshino T, Shirayoshi Y, Higaki K, Messina G, Cossu G, Oshimura M (2009) A highly stable and nonintegrated human artificial chromosome (HAC) containing the 2.4 Mb entire human dystrophin gene. Mol Ther 17:309–317. doi: 10.1038/mt.2008.253 CrossRefGoogle Scholar
- Kakeda M, Nagata K, Osawa K, Matsuno H, Hiratsuka M, Sano A, Okazaki A, Shitara S, Nishikawa S, Masuya A, Hata T, Wako S, Osaki M, Kazuki Y, Oshimura M, Tomizuka K (2011) A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells. Biochem Biophys Res Commun 415:439–444. doi: 10.1016/j.bbrc.2011.10.088 CrossRefGoogle Scholar
- Katoh M, Kazuki Y, Kazuki K, Kajitani N, Takiguchi M, Nakayama Y, Nakamura T, Oshimura M (2010) Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer. BMC Biotechnol 10:37. doi: 10.1186/1472-6750-10-37 CrossRefGoogle Scholar
- Kazuki Y, Hiratsuka M, Takiguchi M, Osaki M, Kajitani N, Hoshiya H, Hiramatsu K, Yoshino T, Kazuki K, Ishihara C, Takehara S, Higaki K, Nakagawa M, Takahashi K, Yamanaka S, Oshimura M (2010) Complete genetic correction of iPS cells from duchenne muscular dystrophy. Mol Ther 18:386–393. doi: 10.1038/mt.2009.274 CrossRefGoogle Scholar
- Kazuki Y, Hoshiya H, Takiguchi M, Abe S, Iida Y, Osaki M, Katoh M, Hiratsuka M, Shirayoshi Y, Hiramatsu K, Ueno E, Kajitani N, Yoshino T, Kazuki K, Ishihara C, Takehara S, Tsuji S, Ejima F, Toyoda A, Sakaki Y, Larionov V, Kouprina N, Oshimura M (2011) Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Ther 18:384–393. doi: 10.1038/gt.2010.147 CrossRefGoogle Scholar
- Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, Barrett JC, Oshimura M, Masumoto H, Earnshaw WC, Larionov V, Kouprina N (2011) Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proc Natl Acad Sci USA 108:20048–20053. doi: 10.1073/pnas.1114483108 CrossRefGoogle Scholar
- Kouprina N, Earnshaw WC, Masumoto H, Larionov V (2013) A new generation of human artificial chromosomes for functional genomics and gene therapy. Cell Mol Life Sci 70:1135–1148Google Scholar
- Ren X, Katoh M, Hoshiya H, Kurimasa A, Inoue T, Ayabe F, Shibata K, Toguchida J, Oshimura M (2005) A novel human artificial chromosome vector provides effective cell lineage-specific transgene expression in human mesenchymal stem cells. Stem Cells 23:1608–1616. doi: 10.1634/stemcells.2005-0021 CrossRefGoogle Scholar
- Shinohara T, Tomizuka K, Miyabara S, Takehara S, Kazuki Y, Inoue J, Katoh M, Nakane H, Iino A, Ohguma A, Ikegami S, Inokuchi K, Ishida I, Reeves RH, Oshimura M (2001) Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down’s syndrome. Hum Mol Genet 10:1163–1175. doi: 10.1093/hmg/10.11.1163 CrossRefGoogle Scholar
- Tomizuka K, Shinohara T, Yoshida H, Uejima H, Ohguma A, Tanaka S, Sato K, Oshimura M, Ishida I (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 97:722–727. doi: 10.1073/pnas.97.2.722 CrossRefGoogle Scholar