BAC TG-EMBED: one-step method for high-level, copy-number-dependent, position-independent transgene expression.
Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.
Bian
Qian
Q
Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL, USA.
Belmont
Andrew S
AS
D000818:Animals; D002843:Chromatin / Q000737:chemistry; D055012:Chromosomal Position Effects; D022202:Chromosomes, Artificial, Bacterial; D033481:Chromosomes, Mammalian; D018628:Gene Dosage; D015870:Gene Expression; D017930:Genes, Reporter; D051379:Mice; D041681:NIH 3T3 Cells; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D019076:Transgenes
D002843:Chromatin; D013762:Tetrahydrofolate Dehydrogenase
In Vitro Cell Dev Biol. 1989 Aug;25(8):697-704
2768131
Cell. 1987 Dec 24;51(6):975-85
3690667
Hum Mol Genet. 1998 Dec;7(13):2079-88
9817925
Curr Opin Genet Dev. 1994 Apr;4(2):281-91
8032206
Blood. 2002 Nov 1;100(9):3077-86
12384402
Nucleic Acids Res. 2005 Feb 24;33(4):e36
15731329
Oncogene. 2004 Nov 11;23(53):8581-90
15467751
Curr Opin Biotechnol. 2002 Apr;13(2):117-23
11950561
Bioessays. 1996 Jan;18(1):55-62
8593164
Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8794-7
1528894
Blood. 2000 May 15;95(10):3242-9
10807795
Nat Biotechnol. 2003 May;21(5):553-8
12679786
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9150-5
10908661
Nature. 1995 Sep 21;377(6546):209-13
7675106
FEBS Lett. 1995 Dec 27;377(3):290-4
8549740
Blood. 2003 Feb 15;101(4):1603-10
12393478
Gene Ther. 2004 May;11(10):819-28
14985785
Nat Biotechnol. 2004 Nov;22(11):1393-8
15529164
Immunol Lett. 2005 Jan 31;96(2):303-4
15585338
Cell Growth Differ. 1995 May;6(5):541-8
7647037
J Cell Biol. 1996 Dec;135(6 Pt 2):1685-700
8991083
Dis Model Mech. 2009 May-Jun;2(5-6):267-74
19383939
J Biotechnol. 2001 Apr 27;87(1):29-42
11267697
Biophys J. 2005 Dec;89(6):4275-85
16150965
Hum Mol Genet. 2000 Apr 12;9(6):937-43
10767317
Trends Biotechnol. 2006 Mar;24(3):137-42
16460822
Cell. 1993 Aug 13;74(3):505-14
8348617
Genetics. 1997 Nov;147(3):1181-90
9383061
Genes Cells. 2004 Nov;9(11):1043-53
15507116
Genomics. 2003 Sep;82(3):269-79
12906852
J Cell Biol. 2009 Apr 6;185(1):87-100
19349581
BMC Biotechnol. 2005 Jun 03;5:17
15935093
Nat Biotechnol. 1997 Sep;15(9):859-65
9306400
Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):575-80
9012826
J Biotechnol. 2004 Jan 22;107(2):95-105
14711493
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6883-8
12011446
EMBO J. 1996 Jul 15;15(14):3713-21
8670875
Cell. 1997 May 16;89(4):655-67
9160756
Nat Genet. 1998 Jan;18(1):56-9
9425901
Genes Dev. 1998 Sep 15;12(18):2852-62
9744862
FASEB J. 2008 Jul;22(7):2534-40
18359926
J Biol Chem. 1989 Dec 15;264(35):21247-56
2592372
Mol Cell Biol. 1997 Jan;17(1):240-7
8972204
Blood. 1999 Nov 1;94(9):3178-84
10556205
Genes Dev. 2002 Jun 15;16(12):1540-54
12080092