Luc Transgene Research Articles

The RNA‐binding protein RBP45D of Arabidopsis promotes transgene silencing and flowering time

RNA-directed DNA methylation (RdDM) helps to defend plants against invasive nucleic acids. In the canonical form of RdDM, 24-nt small interfering RNAs (siRNAs) are produced by DICER-LIKE 3 (DCL3). The siRNAs are loaded onto ARGONAUTE (AGO) proteins leading ultimately to de novo DNA methylation. Here, we introduce the Arabidopsis thaliana prors1 (LUC) transgenic system, in which 24-nt siRNAs are generated to silence the promoter-LUC construct. A forward genetic screen performed with this system identified, besides known components of RdDM (NRPD2A, RDR2, AGO4 and AGO6), the RNA-binding protein RBP45D. RBP45D is involved in CHH (where H is A, C or T) DNA methylation, and maintains siRNA production originating from the LUC transgene. RBP45D is localized to the nucleus, where it is associated with small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). RNA-Seq analysis showed that in CRISPR/Cas-mediated rbp-ko lines FLOWERING LOCUS C (FLC) mRNA levels are upregulated and several loci differentially spliced, among them FLM. In consequence, loss of RBP45D delays flowering, presumably mediated by the release of FLC levels and/or alternative splicing of FLM. Moreover, because levels and processing of transcripts of known RdDM genes are not altered in rbp-ko lines, RBP45D should have a more direct function in transgene silencing, probably independent of the canonical RdDM pathway. We suggest that RBP45D facilitates siRNA production by stabilizing either the precursor RNA or the slicer protein. Alternatively, RBP45D could be involved in chromatin modifications, participate in retention of Pol IV transcripts and/or in Pol V-dependent lncRNA retention in chromatin to enable their scaffold function.

Marker gene tethering by nucleoporins affects gene expression in plants

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localize at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants.

Methyl-CpG-binding domain protein MBD7 is required for active DNA demethylation in Arabidopsis.

Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (Arabidopsis thaliana) line expressing the Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) and Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenes, we isolated an mbd7 (for methyl-CpG-binding domain protein7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis.

HSI2/VAL1 PHD-like domain promotes H3K27 trimethylation to repress the expression of seed maturation genes and complex transgenes in Arabidopsis seedlings.

BackgroundThe novel mutant allele hsi2-4 was isolated in a genetic screen to identify Arabidopsis mutants with constitutively elevated expression of a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene in Arabidopsis. The hsi2-4 mutant harbors a point mutation that affects the plant homeodomain (PHD)-like domain in HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2)/VIVIPAROUS1/ABI3-LIKE1 (VAL1). In hsi2-4 seedlings, expression of this LUC transgene and certain endogenous seed-maturation genes is constitutively enhanced. The parental reporter line (WTLUC) that was used for mutagenesis harbors two independent transgene loci, KanR and KanS. Both loci express luciferase whereas only the KanR locus confers resistance to kanamycin.ResultsHere we show that both transgene loci harbor multiple tandem insertions at single sites. Luciferase expression from these sites is regulated by the HSI2 PHD-like domain, which is required for the deposition of repressive histone methylation marks (H3K27me3) at both KanR and KanS loci. Expression of LUC and Neomycin Phosphotransferase II transgenes is associated with dynamic changes in H3K27me3 levels, and the activation marks H3K4me3 and H3K36me3 but does not appear to involve repressive H3K9me2 marks, DNA methylation or histone deacetylation. However, hsi2-2 and hsi2-4 mutants are partially resistant to growth inhibition associated with exposure to the DNA methylation inhibitor 5-aza-2′-deoxycytidine. HSI2 is also required for the repression of a subset of regulatory and structural seed maturation genes in vegetative tissues and H3K27me3 marks associated with most of these genes are also HSI2-dependent.ConclusionsThese data implicate HSI2 PHD-like domain in the regulation of gene expression involving histone modifications and DNA methylation-mediated epigenetic mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0293-4) contains supplementary material, which is available to authorized users.

Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery.

Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

A novel HSI2 mutation in Arabidopsis affects the PHD-like domain and leads to derepression of seed-specific gene expression

Two related B3 domain transcriptional repressors, HSI2 (HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2)/VAL1 (VP1/ABI3-LIKE1) and HSL1 (HSI2-LIKE1)/VAL2, function redundantly to repress key transcriptional regulators of seed maturation genes in Arabidopsis thaliana seedlings. Using a forward genetic screen designed to isolate trans-acting mutants that affected expression of a transgene containing the glutathione S-transferase F8 promoter::luciferase (GSTF8::LUC) reporter, we identified a novel HSI2 mutant allele, hsi2-4, that exhibits constitutively elevated luciferase expression while expression of the endogenous GSTF8 transcript remains unchanged. The hsi2-4 lesion was found to be a missense mutation that results in the substitution of a conserved cysteine within the plant homeodomain-like (PHD) motif of HSI2. Microarray analysis of hsi2-4 and hsi2-4 hsl1 mutants indicated that the HSI2 PHD-like domain functions non-redundantly to repress a subset of seed maturation genes, including those that encode AGL15 (AGAMOUS-LIKE15), FUSCA3 (FUS3), cruciferins, cupin family proteins, late-embryogenesis abundant protein, oleosins, 2S albumins and other seed-specific proteins in Arabidopsis seedlings. Many genes that are responsive to this mutation in the HSI2 PHD-like domain are enriched in histone H3 trimethylation on lysine 27 residues (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation analysis showed that sequences of the GSTF8::LUC transgene are enriched in H3K27me3 in a HSI2 PHD domain-dependent manner. These results indicate that the transcriptional repression activity of the HSI2 PHD domain could be mediated, at least in part, by its participation in the deposition of H3K27me3 on the chromatin of specific target genes.

Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3.SRDX protein efficiently repressed the transcription of the RD29A::LUC transgene and endogenous RD29A gene in Arabidopsis. Genome wide expression profiling showed that the chimeric repressor also inhibited the expression of several other genes that contain the designer TALE-target sequence in their promoters. Our data suggest that TALEs can be used to generate chimeric repressors to specifically repress the transcription of genes of interest in plants. This sequence-specific transcriptional repression by direct on promoter effector technology is a powerful tool for functional genomics studies and biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-011-9866-x) contains supplementary material, which is available to authorized users.

Role of FOXA and Sp1 in mitochondrial acylcarnitine carrier gene expression in different cell lines

This study investigates the transcriptional role of the human mitochondrial carnitine/acylcarnitine carrier (CAC) proximal promoter. Through deletion analysis, an activation domain (−334/−80bp) was identified which contains FOXA and Sp1 active sites. The wild-type (but not mutated) −334/−80bp region of the CAC gene conferred 74% LUC transgene activity in HepG2 cells, 17% in HEK293 cells and 14% in SK-N-SH cells as compared to that observed with the entire −1503/+3bp proximal promoter. Overexpression and silencing of FOXA2 or Sp1 in HepG2 cells enhanced and diminished, respectively, LUC activity, CAC transcript and CAC protein. In HEK293 and SK-N-SH cells, which do not contain FOXA1-3, LUC activity was increased by FOXA2 overexpression to a greater extent than in HepG2 cells. Both FOXA2 and Sp1 in HepG2, and only Sp1 in HEK293 and SK-N-SH cells, were found to be bound to the CAC proximal promoter. These results show that FOXA and Sp1 sites in HepG2 cells and only the Sp1 site in HEK293 and SK-N-SH cells have a critical role in the transcriptional regulation of the CAC proximal promoter.

Transcription of the mitochondrial citrate carrier gene: Identification of a silencer and its binding protein ZNF224

In the last few years, we have been functionally characterizing the promoter of the human mitochondrial citrate carrier (CIC). In this study we show that CIC silencer activity extends over 26bp (−595/−569), which specifically bind a protein present in HepG2 cell nuclear extracts. This transcription factor was purified by DNA affinity and identified as ZNF224. Overexpression of ZNF224 decreases LUC transgene activity in cells transfected with a construct containing the CIC silencer region, whereas ZNF224 silencing activates reporter transcription in cells transfected with the same construct. Moreover, overexpression and silencing of ZNF224 diminishes and enhances, respectively, CIC transcript and protein levels. Finally, ZNF224 is abundantly expressed in fetal tissues contrary to CIC. It is suggested that CIC transcriptional repression by ZNF224 explains, at least in part, the low expression of CIC in fetal tissues in which fatty acid synthesis is low.

Murine Maternal Cell Microchimerism: Analysis Using Real-Time PCR and In Vivo Imaging1

In humans, maternal cells are present in the affected tissues of children with inflammatory myopathy, scleroderma, and neonatal lupus. It is unknown if maternal cell microchimerism (MCM) contributes to the pathology of disease. We sought to understand the factors that affect MCM to serve as a baseline for future mechanistic studies. Using a mouse model, we bred female mice transgenic for the luciferase (Luc) reporter gene to wild-type (WT) males. The WT offspring were sacrificed at various postnatal ages. DNA was extracted from multiple organs, and real-time PCR amplification was used to quantify Luc transgene as a marker for maternally derived cells. Sensitivity was one to two transgenic cells per 100,000 WT cells. MCM was noted in 85% of mice and 45% of tissues assayed. The average quantity of MCM was 158 maternal cells per 100,000 neonatal cells. The organs displaying the highest frequency and quantity of MCM were heart and lung (P < 0.001). Postnatal age up to 21 days did not appear to affect levels of MCM (P = 0.47), whereas increasing parity may increase levels of MCM. The data show that MCM is a common occurrence in healthy newborn mice, that it is present in their major organs, and that there are organ specific differences. This may represent differential migration of maternal cells or varying receptivity of specific fetal organs to microchimerism. Pregnancy history appears to play a role in maternal cell trafficking. The role of MCM in pregnancy and disease pathogenesis remains to be elucidated.

Bioluminescence Imaging ofPeriod1Gene Expression in Utero

The use of real-time reporters has accelerated our understanding of gene expression in vivo. This study examined the feasibility of a luciferase-based reporter to image spatiotemporal changes in fetal gene expression in utero. We chose to monitor Period1 (Per1) because it is expressed broadly in the body and plays a role in circadian rhythmicity. Using rats carrying a Per1::luc transgene, we repetitively imaged fetuses in utero throughout gestation. We found that bioluminescence was specific to transgenic pups, increased dramatically on embryonic day 10 (10 days after successful mating), and continued to increase logarithmically until birth. Diurnal fluctuations in Per1 expression were apparent several days prior to birth. These results demonstrate the feasibility of in utero imaging of mammalian gene expression, tracking of fetal gene expression from the same litter, and early detection of mammalian clock gene expression. We conclude that luciferase-based reporters can provide a sensitive, noninvasive measure of in utero gene expression.

997. Immune-Mediated Loss of Transgene Expression in Mouse Liver Following Transposon Delivery

An advantage of plasmid-based vector systems for gene therapy over virus-based is their low immunogenicity. The Sleeping Beauty (SB) transposon system, which has the ability to integrate genes into human chromosomes, is a candidate for gene therapy of mucopolysaccharidoses (MPS), genetic disorders of lysosomal metabolism that require sustained expression of the therapeutic transgene. However, our attempts to achieve long-term expression of human alpha -L- iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in almost complete loss of transgene expression and maintenance by 4 weeks postinjection in both MPS and wild type (WT ) mice. In cyclophosphamide-immune-suppressed MPS I mice, the initial supranormal, 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice (see abstract by Aronovich et al). This suggested that cells that expressed the human therapeutic gene induced immune response and were cleared from the liver of some treated mice.The purpose of this study was to analyze contribution of cell- mediated immune response to the clearance of liver cells expressing foreign transgenes. We used high-pressure (hydrodynamic), tail-vein injection to deliver the transposon vectors pT2/mCAGGS-(hIDUA/hGUSB/Luc)//Ub-SB11 to the liver of WT C57Bl/6 mice. The efficiency of injection was evaluated 24 hr postinjection for transgene expression by either enzyme assay for IDUA or GUSB in plasma or in vivo imaging of luciferase (Luc). Mice were euthanized at 7, 10, 14, 17, 21 and 28 days and livers were extracted. Adjacent 5-μm sections from each liver were analyzed for transgene expression (histochemical staining for GUSB), for the presence of cellular infiltrates (H&E staining), and for the presence of CD4+ and CD8+ lymphocytes (immunohistochemical staining). In addition, the maintenance of the hIDUA, hGUSB and Luc transgene, as well as persistence of the transposon plasmid, were determined by PCR. Our results show the temporal relationship between expression of the therapeutic transgene and induction of immune response in the livers of transgene-treated mice.P.B. Hackett and R.S. McIvor have financial interest in Discovery Genomics, Inc. An advantage of plasmid-based vector systems for gene therapy over virus-based is their low immunogenicity. The Sleeping Beauty (SB) transposon system, which has the ability to integrate genes into human chromosomes, is a candidate for gene therapy of mucopolysaccharidoses (MPS), genetic disorders of lysosomal metabolism that require sustained expression of the therapeutic transgene. However, our attempts to achieve long-term expression of human alpha -L- iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in almost complete loss of transgene expression and maintenance by 4 weeks postinjection in both MPS and wild type (WT ) mice. In cyclophosphamide-immune-suppressed MPS I mice, the initial supranormal, 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice (see abstract by Aronovich et al). This suggested that cells that expressed the human therapeutic gene induced immune response and were cleared from the liver of some treated mice. The purpose of this study was to analyze contribution of cell- mediated immune response to the clearance of liver cells expressing foreign transgenes. We used high-pressure (hydrodynamic), tail-vein injection to deliver the transposon vectors pT2/mCAGGS-(hIDUA/hGUSB/Luc)//Ub-SB11 to the liver of WT C57Bl/6 mice. The efficiency of injection was evaluated 24 hr postinjection for transgene expression by either enzyme assay for IDUA or GUSB in plasma or in vivo imaging of luciferase (Luc). Mice were euthanized at 7, 10, 14, 17, 21 and 28 days and livers were extracted. Adjacent 5-μm sections from each liver were analyzed for transgene expression (histochemical staining for GUSB), for the presence of cellular infiltrates (H&E staining), and for the presence of CD4+ and CD8+ lymphocytes (immunohistochemical staining). In addition, the maintenance of the hIDUA, hGUSB and Luc transgene, as well as persistence of the transposon plasmid, were determined by PCR. Our results show the temporal relationship between expression of the therapeutic transgene and induction of immune response in the livers of transgene-treated mice. P.B. Hackett and R.S. McIvor have financial interest in Discovery Genomics, Inc.

Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration

A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent DeltaRBCS1B: LUC transgene. Sixteen luc(+) (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination.

Real-time analysis of rhythmic gene expression in immortalized suprachiasmatic nucleus cells.

Immortalized cells derived from the suprachiasmatic nucleus (SCN) retain many properties of the SCN including the capacity to generate circadian rhythms. Stably transfected SCN2.2 cells expressing the human c- promoter linked to a luciferase reporter gene ( /luc) were examined for evidence of transgene responses to stimuli known to induce c- expression and of endogenous rhythmic variation. Bioluminescence-reported transgene expression was induced in SCN2.2 /luc cells following stimulation with fetal bovine serum or KCl. SCN2.2 /luc cells showed 24 h rhythms of bioluminescence with a 9- to 19-fold difference between peak and minimum levels. These results demonstrate that the regulation of /luc transgene expression in SCN2.2 cells is similar to that of the endogenous c- gene in the SCN.

Characterization of position-induced spatial and temporal regulation of transgene promoter activity in plants.

Quantitative differences in transgene expression between independent transformants are generally ascribed to different integration sites of the transgene (position effect). The contribution of spatial and temporal changes in transgene promoter activity to these position-induced differences in transgene expression in planta are characterized, using the firefly luciferase (luc) reporter system. The activity of three different promoters (Cauliflower Mosaic Virus (CaMV) 35S, modified CaMV 35S and the promoter of an Arabidopsis thaliana Lipid Transfer Protein gene) was shown to vary not only among independent transformants, but also between leaves on the same plant and within a leaf. The differences in local LUC activity between leaves and within a leaf correlated with differences in local luc mRNA steady-state levels. Imaging of LUC activity in the same leaves over a 50 d period, shows that individual transformants can show different types of temporal regulation. Both the spatial and the temporal type of luc transgene expression pattern are inherited by the next generation. It is concluded that previously reported position-induced quantitative differences in transgene expression are probably an accumulated effect of differences in spatial and temporal regulation of transgene promoter activity.

Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me‐JA but is dependent on MAPKK activity

Recognition of avirulent microbial pathogens activates an oxidative burst leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. A novel transgenic Arabidopsis line containing a gst1:luc transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of Pseudomonas syringae pv. tomato (Pst). Strong engagement of the oxidative burst was dependent on the presence of functional Pst hrpS and hrpA gene products. Experiments employing pharmacological agents suggested that at least two distinct sources, including an NADPH oxidase and a peroxidase-type enzyme, contributed to the generation of redox cues. The analysis of gst1 and pal1 gene expression in nahG, coi1 and etr1 plants suggested that engagement of the oxidative burst and cognate redox signalling functioned independently of salicylic acid, methyl jasmonate and ethylene. In contrast, studies using a panel of protein kinase and phosphatase inhibitors and in-gel kinase assays in these mutant backgrounds suggested that a 48 kDa mitogen-activated protein kinase (MAPK) activity was required for the activation of gst1 and pal1 in response to redox cues. Thus the engagement of a bifurcating redox signalling pathway possessing a MAPK module may contribute both to the establishment of plant disease resistance, and to the development of cellular protectant mechanisms.

Luciferase as a reporter gene for transformation studies in rice ( Oryza sativa L.)

Transformed rice plants of var `TN1' were regenerated from immature embryos following particle bombardment with a construct containing the firefly luciferase gene as a reporter gene and the hygromycin resistance gene as a selectable marker. Expression of the luciferase gene in the presence of the substrate luciferin was visualised in the calli derived from bombarded immature embryos and in the leaves and roots of the regenerated transformed plants using a low light imaging system (luminograph). Embryogenic callus proliferation and plant regeneration were unaffected by luciferin treatment and luminograph screening. The quantitative Luc assay using samples of leaf tissue from the segregating generations gave early information about the homozygous and hemizygous state of the luc transgene.

Gene Expression and Chromatin Organization during Mouse Oocyte Growth

Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.

Hypoxia stimulates human preproendothelin-1 promoter activity in transgenic mice.

Significant elevations in endothelin (ET)-1 levels accompany many diseases, but the underlying regulatory mechanisms are unclear. To investigate the in vivo regulation of human preproendothelin-1 (PPET-1), we examined the activity of the PPET-1 promoter in transgenic mice exposed to hypoxia. Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC) reporter transgenes (approximately 2.5 kb, 138 bp, or none of the 5'-flanking sequences of the PPET-1 gene) were generated. LUC expression was reduced in mice with a truncated 138-bp PPET-1 promoter. Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia (10% O2 for 24 h) increased LUC expression sixfold in pulmonary tissue but only twofold in other tissues. In situ hybridization revealed the strongest transgene expression in the pulmonary vasculature and bronchiolar epithelium. These data are consistent with the hypothesis that hypoxic induction of the PPET-1 gene leads to increased pulmonary production of ET-1 in diseases associated with low O2 tension.

Steroid Hormone Regulation and Tissue-Specific Expression of the Human GnRH Gene in Cell Culture and Transgenic Animals

In order to study the molecular mechanisms involved in the control of GnRH gene expression, the human GnRH gene was cloned and characterized. The gene was expressed in cells obtained from CNS tumors in transgenic mice generated utilizing 1131 bp of 5' flanking GnRH DNA fused to the simian virus 40 large T antigen. We have shown a stimulatory estrogen response element in the human GnRH gene by transient transfection studies. DNase I footprinting and an avidinbiotin DNA binding assay demonstrated that the human GnRH gene bound ER. The GN cell line was found to have nuclear ERs utilizing an 125I estradiol binding study and by in situ hybridization histochemistry. In order to study GnRH expression in vivo, either 5000 or 484 bp of GnRH flanking DNA was fused to the luciferase (Luc) reporter gene, and transgenic mice generated. Expression in the transgenic animals was found in the hypothalamus of animals bearing the -500Luc transgene, but not in animals bearing the -484Luc transgene. The transgenic mice expressing the -5000Luc gene were gonadectomized resulting in a 20-30% increase in hypothalamic Luc expression in the males and a 65% increase in females, while mice who were gonadectomized and replaced with testosterone (males) or E2 (females) showed a 50% decrease in Luc expression over control levels. Thus, these studies present in vitro evidence of E2 modulation of GnRH gene expression and an in vivo model in which sensitive studies of GnRH regulation and expression can be performed.