Conditional gene expression in the respiratory epithelium of the mouse

Transgenic mouse models mediating conditional temporal and spatial regulation of gene expression to the respiratory epithelium were developed utilizing the reverse tetracycline transactivator (rtTA) expressed under the control of SP-C and CCSP promoters. Luciferase activity was detected in the lungs of fetal and adult double transgenic mice but was not detected in other tissues or in single transgenic mice. In adult mice, maximal luciferase activity was detected 16h after the administration of doxycycline in the drinking water, or 2h after the injection of doxycycline. Activation of the transgene was observed after the administration of doxycycline in food pellets. After prolonged exposure to doxycycline, luciferase activity decreased slowly following removal of doxycycline, suggesting the importance of tissue pools which maintained expression of the transgene. In SP-C-rtTA mice, exposure of the pregnant dam to doxycycline induced luciferase activity in fetal lung tissue as early as E10.5. Luciferase activity was maintained in the lung tissue of pups during the period of lactation when the mother received doxycycline in the drinking water. In the CCSP-rtTA mice, luciferase was not detected in the absence of doxycycline. In the SP-C-rtTA mice, luciferase activity was detected in the absence of doxycycline but was enhanced approximately 10-fold by administration of drugs. The SP-C-rtTA and CCSP-rtTA activator mice control the expression of transgenes in the developing and mature respiratory epithelium, and will be useful for the study of gene function in the lung.     

Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

Background  

Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern.     

Conditional gene expression in invertebrate animal models

A mechanistic understanding of biology requires appreciating spatiotemporal aspects of gene expression and its functional implications.Conditional expression allows for (ir)reversible switching of genes on or off,with the potential of spatial and/or temporal control.This provides a valuable complement to the more often used constitutive gene (in)activation through mutagenesis,providing tools to answer a wider array of research questions across biological disciplines.Spatial and/or temporal control are granted primarily by(combinations of) specific promoters,temperature regimens,compound addition,or illumination.The use of such genetic tool kits is particularly widespread in invertebrate animal models because they can be applied to study biological processes in short time frames and on large scales,using organisms amenable to easy genetic manipulation.Recent years witnessed an exciting expansion and optimization of such tools,of which we provide a comprehensive overview and discussion regarding their use in invertebrates.The mechanism,applicability,benefits,and drawbacks of each of the systems,as well as further developments to be expected in the foreseeable future,are highlighted.     

Conditional gene expression in human intracranial xenograft tumors

In an initial effort to determine the effect of expressing potentially therapeutic gene products on the growth properties of glioma tumor xenografts, we describe the development of cell lines that can conditionally express beta-galactosidase (beta-gal). To achieve this, we generated stable cell lines that express the modified tetracycline repressor molecule (rtTA) and the beta-gal gene under control of tetracycline-responsive cis-elements. The resulting cell lines express functional beta-gal following treatment with the tetracycline analog doxycyclin (Dox). These cells were then used to form intracranial tumors after injection into the brain using an implantable guide-screw system. The xenografts were found to express beta-gal when the animals were fed drinking water containing Dox. From these studies, we conclude that the expression of a target gene in a human xenograft growing in the brain of a living mouse can be conditionally regulated.     

DNA methylation-mediated control of Sry gene expression in mouse gonadal development

DNA methylation at CpG sequences is involved in tissue-specific and developmentally regulated gene expression. The Sry (sex-determining region on the Y chromosome) gene encodes a master protein for initiating testis differentiation in mammals, and its expression is restricted to gonadal somatic cells at 10.5-12.5 days post-coitum (dpc) in the mouse. We found that in vitro methylation of the 5'-flanking region of the Sry gene caused suppression of reporter activity, implying that Sry gene expression could be regulated by DNA methylation-mediated gene silencing. Bisulfite restriction mapping and sodium bisulfite sequencing revealed that the 5'-flanking region of the Sry gene was hypermethylated in the 8.5-dpc embryos in which the Sry gene was not expressed. Importantly, this region was specifically hypomethylated in the gonad at 11.5 dpc, while the hypermethylated status was maintained in tissues that do not express the Sry gene. We concluded that expression of the Sry gene is under the control of an epigenetic mechanism mediated by DNA methylation.     

Conditional inactivation of the mouse Hus1 cell cycle checkpoint gene

The Hus1 cell cycle checkpoint protein plays a central role in genome maintenance by mediating cellular responses to DNA damage and replication stress. Targeted deletion of mouse Hus1 results in spontaneous chromosomal abnormalities and embryonic lethality. To study the physiological impact of Hus1 deficiency in adult mice, we generated a conditional Hus1 allele, Hus1(flox), in which exons two and three are flanked by loxP sites. Cre-mediated excision of the loxP-flanked region produces Hus1(Delta2,3), which is capable of encoding only 19 of 281 Hus1 amino acids. Germline homozygosity for Hus1(Delta2,3) resulted in mid-gestational embryonic lethality that was indistinguishable from that caused by an established null allele, Hus1(Delta1n). Hus1 was inactivated in adult mice using a transgenic strain in which Cre is sporadically expressed in a variety of tissues from the Hsp70-1 promoter. Conditional Hus1 knockout mice were produced at unexpectedly low frequency and, unlike control animals, demonstrated limited inactivation of the conditional allele, suggesting that Hus1-deficient cells were at a strong selective disadvantage in adult animals. However, viable conditional Hus1 knockout mice consistently showed the greatest degree of Hus1 inactivation specifically in lung and mammary gland, highlighting varying requirements for Hus1 in different tissues. The novel tools described here hold promise for elucidating how the Hus1-dependent checkpoint mechanism contributes to chromosomal stability, DNA damage responses, and tumor suppression in adult mice.     

Conditional allogrooming in the herb-field mouse

Among members of the family Muridae, the herb-field mouse, Apodemus microps, is unique in that aggression is almost entirely lacking.This species, therefore, is a model organism for experimentalstudies of social behavior without the confounding influenceof aggression. We used video surveillance cameras to assessthe importance of self-grooming and allogrooming in the sociallife of this species. Detailed analysis of individual behavioralsequences using Markov chain methods revealed that self-groomingis a relatively stereotypic, sex-independent activity usually lasting about 8 s. Allogrooming is conditional in the herb-fieldmouse, because it takes the form of a reciprocal strategy,with the differences between nonmatching bouts varying accordingto whether the initiator of allogrooming is male or femaleand whether both interactants are of the same or opposite sex.Our analysis revealed that the exchange of allogrooming bouts between individuals of the same sex is reciprocal, but thatmales allow females to "defect" more often than vice versa,and males groomed females for longer than predicted by thedistribution of individual self-grooming bouts. In those specieswhere the demand for mating by males is far greater than thatoffered by females, in other words where females may selectmates, asymmetry of allogrooming may provide a mechanism forfemales to test the "suitability" of males for mating. It mayalso provide the means for males to stimulate females before mating. Furthermore, allogrooming was the only sex-dependentbehavior of the several tested in our experiment. As such,we suggest that allogrooming is the predominant premating mechanismin this species.     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.     

Complex control of mouse apolipoprotein B gene expression revealed by targeted duplication

An elevated plasma level of apolipoprotein B-containing lipoproteins is a risk factor for atherosclerotic cardiovascular disease. Subtle genetic abnormalities in gene expression including an increased expression of the APOB gene may play an important role in determining overall risk. In an attempt to increase mouse Apob expression, we used gene targeting and duplicated approximately 65 kb of genomic DNA containing the Apob locus in its natural genomic position in mice. While we successfully generated mice carrying the Apob gene duplication, the amount of the total Apob mRNA was not increased in their liver. In the intestine, total Apob mRNA was reduced to half of the wild-type mice. Plasma lipids in the Apob duplication mice were not altered. Expression analyses showed that the proximal Apob gene in the duplicated locus was preferentially expressed in both tissues suggesting a limitation of tissue-specific enhancer function. The previously characterized distant intestinal control element was not duplicated, explaining the unequal ratio of intestinal Apob expression. While the existence of an additional liver-specific enhancer element is unknown, our findings suggest the presence of an additional enhancer outside the duplicated region, and that Apob gene expression is more complicated than previously thought.     

Conditional gene expression by controlling translation with tetracycline-binding aptamers

We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA–metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5′-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.     

Conditional expression of transforming growth factor-alpha in adult mouse lung causes pulmonary fibrosis

To determine whether overexpression of transforming growth factor (TGF)-alpha in the adult lung causes remodeling independently of developmental influences, we generated conditional transgenic mice expressing TGF-alpha in the epithelium under control of the doxycycline (Dox)-regulatable Clara cell secretory protein promoter. Two transgenic lines were generated, and following 4 days of Dox-induction TGF-alpha levels in whole lung homogenate were increased 13- to 18-fold above nontransgenic levels. After TGF-alpha induction, transgenic mice developed progressive pulmonary fibrosis and body weight loss, with mice losing 15% of their weight after 6 wk of TGF-alpha induction. Fibrosis was detected within 4 days of TGF-alpha induction and developed initially in the perivascular, peribronchial, and pleural regions but later extended into the interstitium. Fibrotic regions were composed of increased collagen and cellular proliferation and were adjacent to airway and alveolar epithelial sites of TGF-alpha expression. Fibrosis progressed in the absence of inflammatory cell infiltrates as determined by histology, without changes in bronchiolar alveolar lavage total or differential cell counts and without changes in proinflammatory cytokines TNF-alpha or IL-6. Active TGF-beta in whole lung homogenate was not altered 1 and 4 days after TGF-alpha induction, and immunostaining was not increased in the peribronchial/perivascular areas at all time points. Chronic epithelial expression of TGF-alpha in adult mice caused progressive pulmonary fibrosis associated with increased collagen and extracellular matrix deposition and increased cellular proliferation. Induction of pulmonary fibrosis by TGF-alpha was independent of inflammation or early activation of TGF-beta.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.     

Conditional gene targeting in the kidney

Complete mapping of the genome in a number of organisms provides a challenge for experimental nephrologists to identify potential functions of a vast number of new genes in the kidney. Since knockout technologies have evolved in the early eighties the mouse has become a valuable model organism. Researchers can now artificially eliminate the expression of specific genes in a mammalian organism and examine the phenotype. New developments have emerged that allow investigators to knock out a gene specifically in the kidney. Several kidney-specific promoters provide valuable tools and bacterial artificial chromosome (BAC) based techniques like recombineering will enhance both number and accuracy of new mouse lines with spatially controlled gene expression. In addition to spatial control, tetracycline- or tamoxifen-inducible systems, provide the possibility of influencing the temporal expression pattern of a gene enabling researchers to dissect its functions in adult organisms. Knocking out a gene will continue to be the gold standard for defining the role of a specific gene whereas tissue-specific gene knockdown using RNA interference represents an alternative approach for generating lower-priced and fast loss of function models. In addition to reverse genetic approaches, forward genetic techniques like random mutagenesis in mice continue to evolve and will enhance our understanding of disease mechanisms in the kidney.     

Manipulation of mitochondrial DNA gene expression in the mouse

Mitochondrial dysfunction due to impaired respiratory chain function is increasingly recognized as an important cause of human disease. Mitochondrial disorders are relatively common and have an estimated incidence of 1:10,000 live births. There are more than 100 different point mutations and numerous large rearrangements of mitochondrial DNA (mtDNA; mainly single deletions) that cause human disease. We aimed at obtaining an animal model to study physiological aspects of mtDNA mutation disorders. There are as yet unsolved technical problems associated with transfection of mammalian mitochondria. We therefore choose to manipulate mtDNA expression by targeting of the nuclear gene encoding Tfam. We utilised the cre-loxP recombination system to disrupt Tfam since this system allows manipulation of respiratory chain function in selected mouse tissues. We have found increased cell death or apoptosis induction in both germ line and tissue-specific Tfam knockouts. Our results further suggest that increased production of reactive oxygen species (ROS) is not a prominent feature in cells with impaired mtDNA expression.     

Structure and expression of the mouse prealbumin gene

We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.