Repeat-induced gene silencing in mammals

In both plants1–3 and Drosophila melanogastei4,5. expression from a transgenic locus may be silenced when repeated trans-gene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases6–8. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the loxCre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.

[1]  E. Bresnick,et al.  Dual promoter activation by the human beta-globin locus control region. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P. Vyas,et al.  Analysis of the human alpha-globin gene cluster in transgenic mice. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Miyazaki,et al.  Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Inzé,et al.  Suppression of beta‐1,3‐glucanase transgene expression in homozygous plants. , 1992, The EMBO journal.

[5]  M. Van Montagu,et al.  Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[6]  G. Kollias,et al.  Position-independent, high-level expression of the human β-globin gene in transgenic mice , 1987, Cell.

[7]  A. Jarman,et al.  A major positive regulatory region located far upstream of the human alpha-globin gene locus. , 1990, Genes & development.

[8]  E. Whitelaw,et al.  Age-dependent silencing of globin transgenes in the mouse. , 1996, Nucleic acids research.

[9]  S. Henikoff,et al.  Copy number and orientation determine the susceptibility of a gene to silencing by nearby heterochromatin in Drosophila. , 1996, Genetics.

[10]  F. Alt,et al.  Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Steven Henikoff,et al.  Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila , 1994, Cell.

[12]  Germ-line transformation of mice. , 1986, Annual review of genetics.

[13]  N. Proudfoot,et al.  The developmental regulation of the human ζ-globin gene in transgenic mice employing β-galactosidase as a reporter gene , 1992 .

[14]  E. Whitelaw,et al.  A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene , 1997, Molecular and cellular biology.

[15]  N. Martin,et al.  A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice. , 1989, Genes & development.

[16]  J. Rossignol,et al.  Epimutation of repeated genes in Ascobolus immersus. , 1992, The EMBO journal.

[17]  E. Whitelaw,et al.  The vagaries of variegating transgenes. , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[18]  M. Primig,et al.  Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants , 1989, The EMBO journal.

[19]  P. Meyer,et al.  Differences in DNA-methylation are associated with a paramutation phenomenon in transgenic petunia. , 1993, The Plant journal : for cell and molecular biology.

[20]  R. Hoess,et al.  Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Buratowski,et al.  The basics of basal transcription by RNA polymerase II , 1994, Cell.

[22]  E. Signer,et al.  RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[23]  N. Proudfoot,et al.  Alpha‐thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3′ end processing in the human alpha 2 globin gene. , 1986, The EMBO journal.

[24]  E. Whitelaw,et al.  Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status. , 1996, Nucleic acids research.

[25]  Raymond J MacDonald,et al.  Limited transcription of rat elastase I transgene repeats in transgenic mice. , 1988, Genes & development.

[26]  J. Sedat,et al.  The absence of detectable methylated bases in Drosophila melanogaster DNA , 1982, FEBS letters.

[27]  E. Whitelaw,et al.  Position-dependent variegation of globin transgene expression in mice. , 1995, Proceedings of the National Academy of Sciences of the United States of America.