Regulation of Thy-1 gene expression in transgenic mice

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.     

T cell independent Thy-1 allo-antibody response with the use of transgenic mice

We have introduced a mouse Thy-1.1 gene into the germline of Thy-1.2 mice. The introduced gene was shown to be expressed at very high levels in thymocytes when compared with the endogenous gene. Transgenic thymocytes were shown to evoke a higher than normal primary anti-Thy-1.1 antibody response in plaque-forming cell (PFC) assays. This result suggests that a direct quantitative interaction of the Thy-1 antigen activates the B cell response.     

Differential expression of the human Thy-1 gene in rodent-human somatic cell hybrids [corrected

Thy-1 is a cell surface differentiation marker which shows distinct patterns of tissue-specific expression in different species. In man, the Thy-1 antigen is encoded by chromosome 11. We have examined the regulatory signals determining human Thy-1 expression through serologic analysis of rodent-human somatic cell hybrids retaining human chromosome 11 in which the fusion partners belong to distinct differentiation lineages. Cell surface expression of human Thy-1 was determined by mixed hemadsorption assays with two monoclonal antibodies (mAb), K117 and L127, shown to detect authentic human Thy-1 through analysis of COS-7 monkey kidney cells transfected with a cloned human Thy-1 gene. Three different patterns of human Thy-1 expression were observed when hybrid cells, constructed with different human and rodent cell types, were tested with mAb K117 and L127. Hybrids formed between Thy-1+ human neuroblastoma cells and Thy-1- mouse neuroblastoma cells, or hybrids between Thy-1+ human fibroblasts and the Thy-1- mouse kidney carcinoma, RAG, retain human Thy-1 expression. In contrast, hybrids formed between either Thy-1+ human neuroblastoma cells or Thy-1+ human fibroblasts and Thy-1- mouse L cells lose expression of human Thy-1 even though chromosome 11 is retained. Finally, hybrids formed between Thy-1- human peripheral lymphocytes or a Thy-1- lymphoblastoid B cell line and Thy-1- Chinese hamster fibroblasts begin to express human Thy-1. These studies suggest that both positive and negative trans-acting signals may play a role in the tissue-specific regulation of the human Thy-1 gene.     

The maintenance of methylation-free islands in transgenic mice.

The Thy-1 gene is expressed in a tissue- and stage-specific pattern and has a typical 1.6kb methylation-free island (MFI) covering about 600bp upstream and downstream of the two alternative first exons. By microinjection of a mouse Thy-1.1/human Thy-1 gene into fertilized eggs, we were able to show that the MFI is restored in the transgenic mice. The flanking sequence became methylated, but the MFI remains unmethylated in all tissues of transgenic mice at different developmental stages tested, irrespective of the site of expression of the gene. There is one exception, in extra-embryonal tissues of 14.5 day embryos a small percentage of the islands were methylated. We conclude that maintenance of the MFI is regulated by cis-acting sequences present within the gene, and indicates that the unmethylated state of the islands is consistent with a necessary but not sufficient condition for expression of the gene.     

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.     

Function and regulation of gastrin in transgenic mice: a review.

The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.     

Developmentally regulated and erythroid-specific expression of the human embryonic beta-globin gene in transgenic mice.

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Differential expression of the mouse and human Thy-1 gene in embryonal carcinoma cells.

Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.     

Analysis of the Alternative Promoters that Regulate Tissue-Specific Expression of Human Aromatic l-Amino Acid Decarboxylase

Abstract: Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5' untranslated regions of human aromatic l -amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue-specific manner. To determine the cis -acting element regulating the tissue-specific expression of human AADC, we produced three kinds of transgenic mice harboring 5' flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained −7.0 kb to −30 bp in exon N1, including the entire exon L1; ACN contained −3.6 kb to −30 bp in exon N1; and ACL contained −2.8 kb to −42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part of cis -regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expression only in the kidney.     

Ectopic expression of chloramphenicol acetyltransferase (CAT) in the cerebellum in mice transgenic for a carbonic anhydrase II promoter-CAT construct that is without apparent phenotypic effect

We have developed six transgenic lines of mice with constructs containing presumptive 5' regulatory regions of carbonic anhydrase II (CA II). Four of the lines contained 1,100 bases of the 5' flanking region of the human CA II gene, and two transgenic lines resulted from a construct containing 500 bases of the 5' flanking region of the mouse CA II gene. Tissue-specific expression of the chloramphenicol acetyltransferase (CAT) gene was not obtained in any of the transgenic lines. One of the transgenic lines was found to have high levels of expression of CAT in cerebellum. This expression persisted through multiple generations and was independent of the parental origin of the transgene. On the assumption that the expression was due to the insertion of the transgene in or near a gene expressed normally in cerebellum, homozygous mice were bred for the transgenic insert to see if a mutation might have been induced. Homozygous mice were found and seemed to be normal in all aspects of their phenotype studied. Thus, in this case, neither the insertion of the gene nor the ectopic expression of CAT seemed to be harmful to the animals.     

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.