The human CYP1A1 gene is regulated in a developmental and tissue-specific fashion in transgenic mice

Regulation and expression of human CYP1A1 is demonstrated in transgenic mice. We have developed two transgenic mouse lines. One mouse strain (CYPLucR) carries a functional human CYP1A1 promoter (-1612 to +293)-luciferase reporter gene, and the other strain (CYP1A1N) expresses CYP1A1 under control of the full-length human CYP1A1 gene and 9 kb of flanking regulatory DNA. With CYPLucR(+/-) mice, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and several other aryl hydrocarbon receptor ligands induced hepatocyte-specific luciferase activity. When other tissues were examined, TCDD induced luciferase activity in brain with limited induction in lung and no detectable luciferase activity in kidney. Treatment of CYP1A1N(+/-) mice with TCDD resulted in induction of human CYP1A1 in liver and lung, while mouse Cyp1a1 was induced in liver, lung, and kidney. Although induced CYP1A1/Cyp1a1 could not be detected by Western blot analysis in brains from CYP1A1N(+/-) mice, induction in brain was verified by detection of CYP1A1/Cyp1a1 RNA. The administration of TCDD to nursing mothers to examine the effect of lactational exposure via milk demonstrated prominent induction of luciferase activity in livers of CYPLucR(+/-) newborn pups with limited induction in brain. However, TCDD treatment of adult CYPLucR(+/-) mice led to a 7-10-fold induction of brain luciferase activity. Combined these results indicate that tissue-specific and developmental factors are controlling aryl hydrocarbon receptor-mediated induction of human CYP1A1.     

The murine Cyp1a1 gene is expressed in a restricted spatial and temporal pattern during embryonic development

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.     

Induction of the Cyp1a-1 dioxin-responsive enhancer in transgenic mice.

Cyp1a-1, whose product, aryl hydrocarbon hydroxylase, assists in detoxification of polycyclic aromatic hydrocarbons, is the best characterized of the murine cytochrome P450 genes. The Cyp1a-1 dioxin-responsive enhancer region has been previously analyzed in vitro and found to induce expression of heterologous genes upon exposure of transfected cells to various aromatic hydrocarbons. A 2.58 kbp DNA fragment containing the Cyp1a-1 enhancer elements and promoter region was coupled to the chloramphenicol acetyltransferase (CAT) reporter gene and used to create transgenic mice. CAT assays were performed on tissues harvested from three different lines of transgenic mice following mock-induction or induction using the aromatic hydrocarbon, 3-methylcholanthrene. Basal levels of expression were detected in the spleen and small bowel of non-induced mice, with little or no expression detected in the liver. Treatment with 3-methylcholanthrene increased hepatic expression levels by as much as 10,000-fold. More modest levels of induction were also recorded in the spleen, small bowel, kidney, and lung. The results indicate that the dioxin-responsive enhancer region functions as a strongly inducible promoter in vivo. Differences in the response to induction between male and female mice suggest that Cyp1a-1 expression may be governed in a gender related manner.     

Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.     

Ostrich (Struthio camelus) eggshell matrix contains two different C-type lectin-like proteins. Isolation, amino acid sequence, and posttranslational modifications

Comparative analysis of the human and mouse genomic sequences downstream of the apolipoprotein E gene (APOE) revealed a highly conserved element with previously undefined function. In reporter gene transfection studies, this element which is located approximately 42 kb distal to APOE was found to have silencer activity in a subset of cell lines examined. Analysis of transgenic mice containing a fusion construct linking this distal 631 bp conserved element to a reporter gene comprised of the human APOE gene with its proximal promoter resulted in robust brain expression of the transgenic human apoE mRNA in three independent transgenic lines, supporting the identification of a novel brain controlling region (BCR). Further studies using immunohistochemistry revealed widespread human apoE localization throughout the brains of the BCR-apoE transgenic mice with prominent expression in the cortex and diencephalon. In addition, double-label immunofluorescence performed on brain sections and cultures of primary cortical cells localized human apoE protein to cortical neurons and microglia. These studies demonstrate that comparative sequence analysis is a successful strategy to predict candidate regulatory regions in vivo, although they do not imply that this element controls apoE expression physiologically.     

Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Expression of carbonic anhydrase II (CA II) promoter-reporter fusion genes in multiple tissues of transgenic mice does not replicate normal patterns of expression indicating complexity of CA II regulationin vivo

Although the proximal, 5′ 115 bp of the human carbonic anhydrase II (CA II) gene was sufficient for expression of a reporter gene in some transfected cell lines, we found previously that 1100 bp of this promoter (or 500 bp of the mouse CA II promoter) was not sufficient for expression in transgenic mice. We have now studied the expression of linked reporter genes in mice transgenic for either (1) 11 kb of the human 5′ promoter or (2) 8 kb of the human 5′ promoter with mouse sequences from the first exon, part of the first intron (since a CpG island spans this region), and the 3′ sequences of the gene. Expression was found in both cases, but the tissue specificity was not appropriate for CA II. Although there was a difference in the sensitivity of the assays used, the first construct led to expression in many tissues, while the second construct was expressed only in spleen. These findings indicate considerable complexity of DNA control regions for in vivo CA II expression.     

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.     

A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.     

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression.

To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns.

The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice.

These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.     

Characterization of loricrin regulation in vitro and in transgenic mice

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.     

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 brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice

The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.