Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice

The gene for rat pancreatic elastase I is selectively expressed to high levels in the rat exocrine pancreas. When the cloned rat elastase I gene with 7 kb upstream and 5 kb downstream flanking sequences was introduced into mice by microinjection into fertilized eggs, the gene was expressed in a pancreas-specific manner. In four of five transgenic mice, the level of rat elastase I mRNA in the pancreas was equal to or greater than the normal rat level (10,000 mRNAs per cell) and correlated with the number of integrated gene copies. In nonpancreatic tissues the levels were at least 10³-fold lower, except for expression in the liver of one mouse. Thus transfer of a 23 kb genomic DNA segment containing the rat elastase I gene to a foreign chromosomal location in the mouse can give rise to qualitatively and quantitatively normal expression.     

Tissue-specific expression of a vimentin--desmin hybrid gene in transgenic mice.

We have introduced a hybrid gene, pVVim2, composed of the 5' region of the hamster vimentin gene encoding the head and rod domain of vimentin and the 3' region of the hamster desmin gene encoding the tail domain of desmin, into the germ line of mice by pronuclear injection. RNA and protein analysis of mice transgenic for this construct showed that the pVVim2 gene was expressed at high levels in a developmental and tissue-specific manner. This indicates that the vimentin-derived segment of the fusion gene contains all the regulatory elements required for vimentin-specific expression. Immunohistochemical staining of fibroblast cultures derived from the transgenic mice with antibodies specific for vimentin and desmin demonstrated that the pVVim2 protein is assembled into filaments that co-localize with the endogenous vimentin filaments. The expression of pVVim2 protein in mesenchymal cells does not interfere with the function of vimentin in these cells.     

Tissue-specific expression of theHLA-DRA gene in transgenic mice

Transgenic mice were produced containing a 33 kilobase (kb) DNA fragment encompassing the five exons and all the known regulatory regions of the class IIHLA-DRA gene. The transgene displayed regulated expression [constitutive and interferon- (IFN)- induced] of the human products in most mouse tissues. The tissue distribution of theDRA transgene products more closely resembled that of their mouse homologues, the endogenous H-2 Ea products, than the wider distribution ofDRA products in humans. This was evident in several tissues (endothelia of small vessels, especially those of glomerular capillaries, Kupffer cells, and epithelial cells lining the gastrointestinal tract), known to differentially express class II molecules in the two species. Thus, the wider human specific pattern of expression requires an exact cis/trans complementation which is incompletely reconstituted in transgenic mice, suggesting that human specific cis-acting elements may have arisen during evolution to direct the expression of class II genes to those anatomical regions which usually lack them in the mouse. The only example of aberrant expression of theDRA gene in the present series of transgenic mice was in the dendritic and/or epithelial cells of the thymic cortex, which displayed greately reducedDRA levels in spite of a normal expression of the endogenous Ea molecules.     

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.     

Tissue-specific expression of the human renin gene in transgenic mice

Transgenic mice carrying human renin gene were produced by microinjection of 15 kilobases (kb) DNA molecules with up to 3 kb of 5'-flanking sequence and 1.2 kb of 3'-flanking sequence. The transgenes have been shown to be stably transmitted to progeny. It was revealed by RNase protection assay that the human renin gene in a transgenic mouse is expressed preferentially in the kidney. The human renin RNA was also detected at a small level in a variety of tissues such as brain, heart, lung, pancreas, spleen, stomach, testis, and thymus. The direct radioimmunoassay using a monoclonal antibody specific for the active site of human renin demonstrated the synthesis of human active renin in the transgenic mouse kidney. These results suggest that the human renin gene in the transgenic mouse is regulated in a tissue-specific manner.     

Tissue-specific and developmental expression of human transthyretin gene in transgenic mice

To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.     

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.     

Tissue-specific and expression of porcine growth hormone gene in BAC transgenic mice

One of the primary goals of traditional livestock breeding is to improve growth rate and optimise body size. Growth rate can be significantly increased by integrating a growth hormone (GH) transgene under the control of a ubiquitous promoter, but while such animals do demonstrate increased growth there are also serious deleterious side-effects to the animals health. Here we report the generation and initial characterization of transgenic mice that carried a porcine BAC encoding the porcine GH gene. We show that GH expression is restricted specifically to the pituitary, is associated with elevated IGF-1 levels, and results in growth enhancement. No negative effects to the health of the transgenic animals were detected. This initial characterisation supports the use of BAC pGH transgene in livestock studies.     

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.     

Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development.

CD19 is a B-cell-specific member of the immunoglobulin superfamily expressed from early pre-B-cell development until plasma cell differentiation. In vitro studies demonstrate that the CD19 signal transduction molecule can serve as a costimulatory molecule for activation through other B-lymphocyte cell surface molecules. However, much remains to be known regarding how CD19 functions in vivo and whether CD19 has different roles at particular stages of B-cell differentiation. Therefore, transgenic mice overexpressing the human CD19 (hCD19) gene were generated to determine whether this transgene would be expressed in a B-lineage-specific fashion and to dissect the in vivo role of CD19 in B-cell development and activation. Expression of the human transgene product was specifically restricted to all B-lineage cells and appeared early in development as occurs with hCD19. In addition, expression of hCD19 severely impaired the development of immature B cells in the bone marrow, with dramatically fewer B cells found in the spleen, peripheral circulation, and peritoneal cavity. The level of hCD19 expressed on the cell surface correlated directly with the severity of the defect in different transgenic lines. These results demonstrate that the hCD19 gene is expressed in a lineage-specific fashion in mice, indicating that the hCD19 gene may be useful for mediating B-lineage-specific expression of other transgene products. In addition, these results indicate an important role for the lineage-specific CD19 molecule during early B-cell development before antigen-dependent activation.     

Tissue-specific, high level expression of the rat whey acidic protein gene in transgenic mice.

The importance of intragenic and 3' flanking sequences in the control of the temporal, hormonal and tissue-specific expression of milk whey acidic protein (WAP) has been demonstrated in transgenic mice. Mouse lines carrying a 4.3 kb genomic clone containing the entire rat WAP gene minus 200 bp of the first intron with 0.949 kb of 5' and 1.4 kb of 3' flanking DNA were generated. In eight of nine independent lines of mice analyzed, WAP transgene expression was detected at levels ranging from 1% to 95% (average, 27%) of the endogenous gene. The transgene was expressed preferentially in the mammary gland. Although developmentally regulated during pregnancy and lactation, the temporal pattern of WAP transgene expression differed from the endogenous gene. A precocious increase in expression of the transgene was detected at 7 days of pregnancy, several days earlier in pregnancy than the major increase observed in endogenous mouse WAP mRNA. The rat WAP transgene was translated and secreted into the milk of transgenic mice at levels comparable to the endogenous mouse WAP. This is the first report of a gene that is negatively regulated in dissociated cell cultures as well as in transfected cells, yet is expressed efficiently in the correct multicellular environment of the transgenic mouse.     

Tissue-specific expression of the chicken alpha A-crystallin gene in cultured lens epithelia and transgenic mice

The present experiments show that the single gene for the lens-specific protein alpha A-crystallin of chickens and mice uses a different subset of cis- and trans-acting regulatory elements for expression in transfected embryonic chicken lens epithelial cells. A chicken alpha A-crystallin-chloramphenicol acetyltransferase (CAT) fusion gene required 162 base pairs whereas the murine alpha A-crystallin-CAT fusion gene required only 111 base pairs of 5'-flanking sequences for efficient tissue-specific expression in the transfected chicken lens cells. Gel retardation and competition experiments were performed using embryonic chicken lens nuclear extract and oligodeoxynucleotides identical to the 5'-flanking region of the chicken (-170/-111) and murine (-111/-88 and -88/-55) alpha A-crystallin gene. The results indicated that these homologous promoters use different nuclear factors for function. Methylation interference analysis identified a dyad of symmetry (CTGGTTCCCACCAG) at position -153 to -140 in the chicken alpha A-crystallin promoter which binds one or more lens nuclear factors. Gel mobility shift experiments using nuclear extracts of brain, reticulocytes, and muscle of embryonic chickens or HeLa cells suggested that the factor(s) binding to the chicken alpha A-crystallin gene promoter sequences are not lens specific. Despite differences in the functional and protein-binding properties of the alpha A-crystallin gene promoter of chickens and mice, expression of the chicken alpha A-crystallin-CAT fusion gene in transgenic mice was lens specific, consistent with a common underlying mechanism for expression of the alpha A-crystallin gene in chickens and mice.     

Tissue-specific expression of a wheat high molecular weight glutenin gene in transgenic tobacco.

The expression of a wheat genomic clone containing the entire coding sequence of the high molecular weight glutenin subunit 12 gene flanked by 2.6 kilobases of 5' and 1.5 kilobases of 3' sequences has been studied after introduction into tobacco. Seeds of different tobacco plants containing the full-length wheat genomic clone accumulated different amounts of intact high molecular weight glutenin subunit mRNA and of a polypeptide displaying the solubility, molecular weight, and antigenic properties of the high molecular weight glutenin subunit 12. The wheat protein accumulated without obvious degradation products and constituted up to approximately 0.1% of the total tobacco endosperm protein. Restriction fragments corresponding to 2.6 kilobases, 1.4 kilobases, and 433 base pairs of high molecular weight glutenin 5' upstream sequence were fused to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene in the vector polyCATter and transferred into tobacco. Chloramphenicol acetyltransferase enzyme activity was detected only in the seed endosperm tissue of the transformed plants. It was detected in tobacco seeds 8 days after anthesis and persisted until seed maturity. It is concluded that 433 base pairs of high molecular weight glutenin upstream sequence are sufficient to confer endosperm-specific expression of this monocot gene in the dicot tobacco.     

Tissue-specific methylation of a CpG island in transgenic mice.

Clustering of CpG dinucleotides in CpG-rich islands is a characteristic feature of mammalian genomes. Such CpG islands are frequently associated with genes and usually hypomethylated, regardless of the gene activity. This is the case for the CpG island of the murine Thy-1 gene. A transgenic line containing multiple copies of a truncated, concatemeric CpG island from the Thy-1.1 allele (Thy-1.2 background) showed that a stable fraction (approx. 0.20) became fully methylated in somatic tissues of homozygous mice with respect to testable restriction sites, while the remaining copies were methylation-free, i.e., this methylation appears to be an 'all-or-none' phenomenon. DNA from extraembryonic tissues (placenta and yolk sac) and epididymal sperm showed, however, an even higher degree of methylation in two distinct patterns. In the extraembryonic tissue, partial methylation of each copy was seen, whereas in sperm a high degree of 'all-or-none' methylation (greater than 0.35) was observed.