Identification and expression of cosmids with an allelic variant of class I alcohol dehydrogenase in transgenic mice

The mouse Adh1 gene exhibits tissue-specific regulation, is developmentally regulated, and is androgen regulated in kidney and adrenal tissue. To study this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper tissue expression and hormonal control. Transgenic mice have been produced with an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5′-flanking sequence [1]. Complete androgen regulation in kidney requires a region between −2.5 and −10 kb. A sequence extending to −10 kb does not confer liver expression in this minigene construct. B6.S mice express an electrophoretically variant protein resulting from a known nucleotide substitution resulting in a restriction endonuclease length polymorphism. Transgenic mice harboring B6.S cosmids can be studied for expression analysis at both protein and mRNA levels, identification of transgenic founders and inheritance studies are greatly facilitated by a PCR-restriction endonuclease cleavage approach, the entire mouse gene is used as a reporter, and the formation of heterodimeric enzyme molecules can be used to infer expression of the transgene in the proper cell types within a given tissue. Expression of a B6.S cosmid containing the entire Adh1 gene and 6 kb of 5′- and 21 kb of 3′-flanking region occurs in transgenic mice in a copy number dependent manner in a number of tissues, but expression in liver does not occur. The ability to analyze expression at the protein and mRNA levels has been confirmed using this system. Future directions will involve the use of large BAC clones modified by RARE cleavage to identify the liver specific elements necessary for expression.     

Distal and proximal cis-linked sequences are needed for the total expression phenotype of the mouse alcohol dehydrogenase 1 (Adh1) gene

Mouse alcohol dehydrogenase 1 (Adh1) gene expression occurs at high levels in liver and adrenal, moderate levels in kidney and intestine, low levels in a number of other tissues, and is undetectable in thymus, spleen and brain by Northern analysis. In transgenic mice, a minigene construct containing 10 kb of upstream and 1.5 kb of downstream flanking sequence directs expression in kidney, adrenal, lung, epididymis, ovary and skin but promotes ectopic expression in thymus and spleen while failing to control expression in liver, eye, intestine and seminal vesicle. Cosmids containing either 7 kb of upstream and 21 kb of downstream or 12 kb of upstream and 23 kb of downstream sequence flanking genetically marked Adh1 additionally promotes seminal vesicle expression suggesting downstream or intragenic sequence controls expression in this tissue. However, expression in liver, adrenal, or intestine is not promoted. The Adh1(a) allele on the cosmid expresses an enzyme electrophoretically distinct from that of the endogenous Adh1(b) allele, and presence of the heterodimeric enzyme in expressing tissues confirms that transgene activity occurs in the same cell-type as the endogenous gene. Transgene expression levels promoted by cosmids were at physiologically relevant amounts and exhibited greater copy-number dependence than observed with minigenes. Transgene mRNA expression correlated with expression measured at the enzyme level. A bacterial artificial chromosome containing 110 kb of 5'- and 104 kb of 3'-flanking sequence surrounding the Adh1 gene promoted expression in tissues at levels comparable to the endogenous gene most importantly including liver, adrenal and intestinal tissue where high level Adh1 expression occurs. Transgene expression in liver was in the same cell types as promoted by the endogenous gene. Although proximal elements extending 12 kb upstream and 23 kb downstream of the Adh1 gene promote expression at physiologically relevant levels in most tissues, more distal elements are additionally required to promote normal expression levels in liver, adrenal and intestinal tissue where Adh1 is most highly expressed.     

The mitochondrial uncoupling protein gene in brown fat: correlation between DNase I hypersensitivity and expression in transgenic mice.

The mitochondrial uncoupling protein gene is rapidly induced in mouse brown fat following cold exposure. To identify cis-regulatory elements, approximately 50 kb of chromatin surrounding the uncoupling protein gene was examined for its hypersensitivity to DNase I. Seven DNase I-hypersensitive sites were identified in the 5'-flanking DNA, and one site was identified in the 3'-flanking DNA. Transgenic mice with an uncoupling protein minigene were generated by microinjection of fertilized eggs with a transgene containing 3 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. Expression of the transgene is restricted to brown fat and is cold inducible. Four additional transgenic lines were generated with a second transgene containing a 1.8-kb deletion in the 5'-flanking DNA, and expression of this minigene is absent in all tissues analyzed. A DNase I-hypersensitive site located in the 1.8-kb deletion contains a cyclic AMP response element that binds a brown fat tumor enriched nuclear factor. On the basis of these observations, we propose that a cis-acting regulatory sequence between -3 and -1.2 kb of the 5'-flanking region, possibly at a DNase I-hypersensitive site, is required for controlling uncoupling protein expression in vivo.     

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5' flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue-specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co-express the transgenes in hepatocytes were obtained. Hepatoma-derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.     

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.     

Two 5'-regions are required for nutritional and insulin regulation of the fatty-acid synthase promoter in transgenic mice

We previously reported that 2.1 kilobase pairs of the 5'-flanking sequence are sufficient for tissue-specific and hormonal/metabolic regulation of the fatty-acid synthase (FAS) gene in transgenic mice. We also demonstrated that the -65 E-box is required for insulin regulation of the FAS promoter using 3T3-L1 adipocytes in culture. To further define sequences required for FAS gene expression, we generated transgenic mice carrying from -644, -444, -278, and -131 to +67 base pairs of the rat FAS 5'-flanking sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Similar to the expression observed with -2100-FAS-CAT transgenic mice, transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT expressed high levels of CAT mRNA only in lipogenic tissues (liver and adipose tissue) in a manner identical to the endogenous FAS mRNA. In contrast, -278-FAS-CAT and -131-FAS-CAT transgenic mice did not show appreciable CAT expression in any of the tissues examined. When previously fasted mice were refed a high carbohydrate, fat-free diet, CAT mRNA expression in transgenic mice harboring -644-FAS-CAT and -444-FAS-CAT was induced dramatically in liver and adipose tissue. The induction was virtually identical to that observed in -2100-FAS-CAT transgenic mice and to the endogenous FAS mRNA. In contrast, -278-FAS-CAT transgenic mice showed induction by feeding, but at a much lower magnitude in both liver and adipose tissue. The -131-FAS-CAT transgenic mice did not show any CAT expression either when fasted or refed a high carbohydrate diet. To study further the effect of insulin, we made these transgenic mice insulin-deficient by streptozotocin treatment. Insulin administration to the streptozotocin-diabetic mice increased CAT mRNA levels driven by the -644 FAS and -444 FAS promoters in liver and adipose tissue, paralleling the endogenous FAS mRNA levels. In the case of -278-FAS-CAT, the induction observed was at a much lower magnitude, and deletion to -131 base pairs did not show any increase in CAT expression by insulin. This study demonstrates that the sequence requirement for FAS gene regulation employing an in vitro culture system does not reflect the in vivo situation and that two 5'-flanking regions are required for proper nutritional and insulin regulation of the FAS gene. Cotransfection of the upstream stimulatory factor and various FAS promoter-luciferase constructs as well as in vitro binding studies suggest a function for the upstream stimulatory factor at both the -65 and -332 E-box sequences.     

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.     

Human CD2 3'-flanking sequences confer high-level, T cell-specific, position-independent gene expression in transgenic mice

We have localized a set of T cell-specific DNAase I hypersensitive sites in the 3'-flanking region of the human CD2 gene. A 5.5 kb BamHI-XbaI fragment containing these DNAase I hypersensitive sites conferred efficient, copy number-dependent, T cell-specific expression of a linked human CD2 minigene, independent of the position of integration in the transgenic mouse genome. When linked to the mouse Thy-1.1 gene or the human beta-globin gene, this fragment conferred the same T cell-specific expression, independent of its orientation. These results suggest that this flanking region is both necessary and sufficient for full tissue-specific activation of homologous and heterologous genes in transgenic mice.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

Transgenic mice selectively lacking MHC class II (I-E) antigen expression on B cells: an in vivo approach to investigate Ia gene function

The E alpha MHC class II gene with 1.4 kb of 5'-flanking and 0.5 kb of 3'-flanking sequences was introduced into (H-2b X s)F2 mice, which do not express their endogenous E alpha gene. The transgene was expressed in thymic tissue and in adherent spleen cells and was induced in peritoneal exudate cells by gamma-interferon. In contrast to the normal E alpha gene, there was no expression in B lymphocytes. Since transgenic animals made with constructs containing 3.2 kb and 2 kb of 5'-flanking sequences show normal expression pattern of the E alpha gene, it appears that deletion of 5'-flanking sequences between -1.4 kb and -2 kb inactivated or eliminated regulatory sequences required for expression of E alpha specifically in B cells. The presence of pBR327 DNA linked to the -1.4 kb E alpha transgene suppresses expression in peripheral adherent cells, yielding mice expressing E alpha only in the thymus. These mice appear to be tolerant to I-E, as measured in mixed leukocyte response experiments.     

Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22 584–22 588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2 kb genomic DNA fragment 5′ of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, and β-geo hybrid reporter gene. The β-galactosidase (βGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, βGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial–temporal expression patterns of the βGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong βGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, βGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2 kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of βGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.     

Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.     

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.     

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml⁻¹) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml⁻¹. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.     

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.