title: Enucleation of feeder cells and egg cells with psoralens.
abstract: The cell nucleus must be inactivated or destroyed in order to generate feeder layers for cultured cells or to prepare recipient egg cells for nuclear transfer. Existing enucleation techniques are either cumbersome or employ toxic chemicals. Here we report a new method to enucleate cells by treatment with a psoralen and long-wave ultraviolet light. The technique is >90% efficient and causes little cytoplasmic damage to the treated cell. We have used psoralen treatment to enucleate a wide variety of cells, including eggs, sperm, HeLa cells, and fibroblasts. Colonies of human embryonic stem cells (hESCs) and human keratinocyte precursors grown on psoralen-treated feeders are indistinguishable from those grown on gamma-irradiated or mitomycin C-treated cells. Psoralen enucleation provides a rapid, simple, and non-toxic method to generate feeder cells. The technique is also useful for nuclear transfer studies in species with large eggs whose cleavage divisions are not regulated by cell-cycle checkpoints.
authors.affiliation: Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. t-mcgarry@northwestern.edu
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McGarry
authors.forename: Michael
authors.initials: M
authors.lastname: Bonaguidi
authors.forename: Ljuba
authors.initials: L
authors.lastname: Lyass
authors.forename: John A
authors.initials: JA
authors.lastname: Kessler
authors.forename: Jason M
authors.initials: JM
authors.lastname: Bodily
authors.forename: Lynn
authors.initials: L
authors.lastname: Doglio
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D005260:Female; D011564:Furocoumarins / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007621:Karyotyping; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D017319:Photosensitizing Agents / Q000494:pharmacology*; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays; D014982:Xenopus laevis / Q000196:embryology / Q000254:growth & development
chemical_list: D011564:Furocoumarins; D017319:Photosensitizing Agents
references.citation: Science. 2005 Aug 26;309(5739):1369-73
references.pmid: 16123299
references.citation: J Cell Biol. 1988 Dec;107(6 Pt 1):2009-19
references.pmid: 3143733
references.citation: Science. 1994 Oct 28;266(5185):650-3
references.pmid: 7939720
references.citation: Cloning Stem Cells. 2003;5(4):379-87
references.pmid: 14733755
references.citation: Cloning Stem Cells. 2004;6(1):5-13
references.pmid: 15107241
references.citation: Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14389-93
references.pmid: 10588715
references.citation: Mol Reprod Dev. 1993 Apr;34(4):427-30
references.pmid: 8471262
references.citation: J Gen Virol. 2002 Nov;83(Pt 11):2753-2763
references.pmid: 12388811
references.citation: Development. 1993 Oct;119(2):447-56
references.pmid: 8287796
references.citation: Reprod Fertil Dev. 2005;17(8):791-7
references.pmid: 16476206
references.citation: J Cell Biol. 1984 Apr;98(4):1247-55
references.pmid: 6425302
references.citation: Stem Cells Dev. 2009 May;18(4):641-51
references.pmid: 18764735
references.citation: N Engl J Med. 2007 Aug 16;357(7):682-90
references.pmid: 17699818
references.citation: Curr Biol. 2004 Jan 6;14(1):R35-45
references.pmid: 14711435
references.citation: Science. 1998 Nov 6;282(5391):1145-7
references.pmid: 9804556
references.citation: Genes Dev. 2000 Jun 15;14(12):1448-59
references.pmid: 10859164
references.citation: Mol Reprod Dev. 2001 Apr;58(4):432-6
references.pmid: 11241780
references.citation: Nat Biotechnol. 2000 Feb;18(2):223-5
references.pmid: 10657133
references.citation: Genes Dev. 2000 Jun 15;14(12):1439-47
references.pmid: 10859163
references.citation: Biol Reprod. 1995 Nov;53(5):1088-94
references.pmid: 8527512
references.citation: Methods Cell Biol. 1991;36:299-309
references.pmid: 1811139
references.citation: J Androl. 2000 Sep-Oct;21(5):610-5
references.pmid: 10975406
references.citation: Cloning Stem Cells. 2006 Winter;8(4):241-50
references.pmid: 17196089
references.citation: Methods Cell Biol. 1991;36:581-605
references.pmid: 1839804
references.citation: Cytogenetics. 1972;11(4):270-8
references.pmid: 5072789
references.citation: EMBO J. 2004 Mar 10;23(5):1178-87
references.pmid: 14988723
references.citation: Annu Rev Biochem. 1985;54:1151-93
references.pmid: 2411210
references.citation: J Reprod Fertil. 1993 Sep;99(1):39-44
references.pmid: 8283450
references.citation: Curr Protoc Cytom. 2001 May;Chapter 7:Unit 7.5
references.pmid: 18770732