Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.     

Accumulation of human apolipoprotein E in the plasma of transgenic mice

Three separate lines of transgenic mice were created with integrated copies of an 11.1-kilobase pair human DNA fragment containing the apolipoprotein (apo) E gene. The endogenous mouse apoE gene is primarily expressed in the liver with varying levels of expression in other tissues. However, in all three transgenic lines high levels of human apoE mRNA were detected only in the kidney, with lower levels found in the liver and other tissues; despite this profile of human apoE mRNA, human apoE was found in the plasma of the transgenic mice at levels comparable to those found in human plasma. All of the human apoE in the plasma of the transgenic mice was associated with lipoproteins. These results suggest that the domain responsible for the high level of apoE expression in liver lies outside of the microinjected DNA fragment and that an ectopic site of expression of an introduced gene may be permissive for the accumulation of its protein in plasma.     

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice.

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.     

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.     

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.     

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.     

apoE_4近交系转基因鼠的高脂血症表现和自发变换行为损害

建立相关疾病的动物模型 ,研究apoE4在脂质代谢和早老性痴呆等疾病中的作用 .通过显微注射法建立人apoE4近交系转基因鼠 .经Southern和Northern印迹杂交 ,鉴定apoE4基因的整合与表达 .98只新生鼠中鉴定出 2只首建鼠 ,定名为TgN(apoE4) 1QiL和TgN(apoE4) 2QiL .外源基因整合的拷贝数分别为 1和 2 .F1代杂合鼠的脑 ,肾脏 ,心脏和肝脏中均有人apoE4基因的表达 .血清脂质水平通过酶法检测 ,自发变换行为经Y迷宫试验检测 .转基因鼠的血清胆固醇和甘油三酯明显升高 ,自发变换行为受到损害 .结果表明 ,近交系转基因鼠过量表达人apoE4基因可导致血清脂质升高 ,并对其空间记忆能力造成损害 .     

Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.     

Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice.

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.     

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.     

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.     

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.     

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.     

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.