Altering the Expression in Mice of Genes by Modifying Their 3 (cid:1) Regions

previously developed a “gene titration” method in which homologous recombinationin stem(ES) cells is usedto deleteor duplicatea chosengene at itsnormal chromosomal location without the sequence of the gene product of nearby cis -acting control ele-Masao the ES cells modified in this way have one, two (wild- type), three, or four functional copies of the gene of interest. A linear and strictly proportional relationship between the number of copies of a gene and the steady-state level of its product was observed in mice resulting from several experiments of this type (Krege et al., 1997; Summary Oliver al., 1998). This agrees with previous observations in humans that heterozygotes for a completely Polymorphic differences altering expression of genes nonfunctional mutation usually have close to 50% nor- without changing their products probably underlie hu-mal levels of gene product (Harris, 1970), while trisomic man quantitative traits affecting risks of serious dis-individuals have close to 150% normal levels (Epstein, eases, but methods for investigating such quantitative 1989).Fifty percentexpressionis consequentlyregularly differences in animals are limited. Accordingly, we observed at the mRNA level in mice heterozygous for a have developed a procedure for changing the expres-gene deletion “knock out.” However, in the three- and sion in mice of chosen genes over a 100-fold range four copy animals, the chromosome with the duplicated while retaining their chromosomal Region for a Target Gene fluorescence of the modified ES cells for seven of our In order to design a modification to obtain a desired tested series of 3 (cid:1) regions with that from cardiomyo-change in the expression of a chosen target gene, it is cytes and trophoblastocytes derived from these ES necessary to compare the “strength” of the natural cells. The rank order of the seven 3 (cid:1) regions in the parent 3 (cid:1) UTR and GRE from the target gene with that of pre- ES cells and in the two types of differentiated cells gen-viously tested 3 (cid:1) regions. We illustrate this aspect of our erated from them is identical.

[1]  Helen V. Firth,et al.  Down syndrome (trisomy 21) , 2005 .

[2]  S. John,et al.  Molecular phenotyping for analyzing subtle genetic effects in mice: Application to an angiotensinogen gene titration , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Kiledjian,et al.  Functional Link between the Mammalian Exosome and mRNA Decapping , 2001, Cell.

[4]  International Human Genome Sequencing Consortium.  Initial sequencing and analysis of the human genome , 2001, Nature.

[5]  Satoshi Tanaka,et al.  PPARγ Mediates High-Fat Diet–Induced Adipocyte Hypertrophy and Insulin Resistance , 1999 .

[6]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[7]  M. F. Goy,et al.  Natriuretic peptide receptor 1 expression influences blood pressures of mice in a dose-dependent manner. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[8]  H. S. Kim,et al.  Angiotensin-converting enzyme gene mutations, blood pressures, and cardiovascular homeostasis. , 1997, Hypertension.

[9]  J. Ross,et al.  mRNA stability in mammalian cells. , 1995, Microbiological reviews.

[10]  H. S. Kim,et al.  Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[11]  C. Malbon,et al.  The M(r) 35,000 beta-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition. , 1993, The Journal of biological chemistry.

[12]  T. Doetschman,et al.  Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Ronald W. Davis,et al.  The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation , 1989, Cell.

[14]  D. Mccormick.  Sequence the Human Genome , 1986, Bio/Technology.

[15]  G. Shaw,et al.  A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation , 1986, Cell.