24-脱氢胆固醇还原酶基因转基因小鼠血生化指标分析

目的建立全身表达24-脱氢胆固醇还原酶基因（Dhcr24）转基因小鼠动物模型,研究该基因过表达对小鼠代谢的影响。方法RT-PCR法克隆小鼠Dhcr24基因,把该基因插入CMV启动子下游,构建转基因表达载体,通过显微注射法建立Dhcr24转基因小鼠。PCR鉴定Dhcr24转基因小鼠的基因型,RT-PCR和Western Blot检测基因表达水平,血生化检测仪检测转基因小鼠血生化指标的改变。结果建立了2个不同表达水平的Dhcr24转基因小鼠品系,转入的Dhcr24基因在肝和脾组织中的表达高于内源的Dhcr24。血生化检测证实：乳酸脱氢酶（LDH）、总蛋白（TP）、白蛋白（Alb）和血肌酐（SCr）较野生型小鼠明显降低,而高密度脂蛋白胆固醇（HDL-c）和碱性磷酸酶（ALP）较野生型小鼠明显增加,并且Dhcr24转基因雌鼠的体重比野生型小鼠明显降低,均有显著差异。但Dhcr24转基因雄鼠各项指标与野生型小鼠相比没有显著差异。结论成功建立了全身表达Dhcr24转基因小鼠,并证实Dhcr24基因对雌性小鼠的体重和血生化指标,包括LDH,TP,Alb,SCr,HDL-c and ALP具有明显的影响。     

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene

Key message

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ).

Abstract

Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T₁ generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T₀ lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.     

Differential mRNA expression of seven genes involved in cholesterol metabolism and transport in the liver of atherosclerosis-susceptible and -resistant Japanese quail strains

Background

Two atherosclerosis-susceptible and -resistant Japanese quail (Coturnix japonica) strains obtained by divergent selection are commonly used as models to study atherosclerosis, but no genetic characterization of their phenotypic differences has been reported so far. Our objective was to examine possible differences in the expression of genes involved in cholesterol metabolism and transport in the liver between these two strains and to evaluate the value of this model to analyze the gene system affecting cholesterol metabolism and transport.

Methods

A factorial study with both strains (atherosclerosis-susceptible versus atherosclerosis-resistant) and two diets (control versus cholesterol) was carried out. The mRNA concentrations of four genes involved in cholesterol biosynthesis (HMGCR, FDFT1, SQLE and DHCR7) and three genes in cholesterol transport (ABCG5, ABCG8 and APOA1) were assayed using real-time quantitative PCR. Plasma lipids were also assayed.

Results

Expression of ABCG5 (control diet) and ABCG8 (regardless of dietary treatment) and expression of HMGCR, FDFT1 and SQLE (regardless of dietary treatment) were significantly higher in the atherosclerosis-resistant than in the atherosclerosis-susceptible strain. Plasma triglyceride and LDL levels, and LDL/HDL ratio were significantly higher in the atherosclerosis-susceptible than in the atherosclerosis-resistant strain fed the cholesterol diet. In the atherosclerosis-susceptible strain, ABCG5 expression regressed significantly and positively on plasma LDL level, whereas DHCR7 and SQLE expression regressed significantly and negatively on plasma triglyceride level.

Conclusions

Our results provide support for the hypothesis that the atherosclerosis-resistant strain metabolizes and excretes cholesterol faster than the atherosclerosis-susceptible strain. We have also demonstrated that these quail strains are a useful model to study cholesterol metabolism and transport in relation with atherosclerosis.     

Overexpression of miR-155 in the Liver of Transgenic Mice Alters the Expression Profiling of Hepatic Genes Associated with Lipid Metabolism

Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.     

Cosuppression of RBCS3B in Arabidopsis leads to severe photoinhibition caused by ROS accumulation

Key message

Cosuppression of an Arabidopsis Rubisco small subunit gene RBCS3B at Arabidopsis resulted in albino or pale green phenotypes which were caused by ROS accumulation

Abstract

As the most abundant protein on Earth, Rubisco has received much attention in the past decades. Even so, its function is still not understood thoroughly. In this paper, four Arabidopsis transgenic lines (RBCS3B-7, 18, 33, and 35) with albino or pale green phenotypes were obtained by transformation with a construct driving expression of sense RBCS3B, a Rubisco small subunit gene. The phenotypes produced in these transgenic lines were found to be caused by cosuppression. Among these lines, RBCS3B-7 displayed the most severe phenotypes including reduced height, developmental arrest and plant mortality before flowering when grown under normal light on soil. Chloroplast numbers in mesophyll cells were decreased compared to WT, and stacked thylakoids of chloroplasts were broken down gradually in RBCS3B-7 throughout development. In addition, the RBCS3B-7 line was light sensitive, and PSII activity measurement revealed that RBCS3B-7 suffered severe photoinhibition, even under normal light. We found that photoinhibition was due to accumulation of ROS, which accelerated photodamage of PSII and inhibited the repair of PSII in RBCS3B-7.     

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice

Background

In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development.

Results

We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes.

Conclusion

We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression.     

Genetic replacement of surfactant protein-C reduces respiratory syncytial virus induced lung injury

Background

Individuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV.

Methods

In order to determine requirements for correction of RSV induced injury we have generated compound transgenic mice where SP-C expression can be induced on the Sftpc -/- background (SP-C/Sftpc -/-) by the administration of doxycycline (dox). The pattern of induced SP-C expression was determined by immunohistochemistry and processing by Western blot analysis. Tissue and cellular inflammation was measured following RSV infection and the RSV-induced cytokine response of isolated Sftpc +/+ and -/- type II cells determined.

Results

After 5 days of dox administration transgene SP-C mRNA expression was detected by RT-PCR in the lungs of two independent lines of bitransgenic SP-C/Sftpc -/- mice (lines 55.3 and 54.2). ProSP-C was expressed in the lung, and mature SP-C was detected by Western blot analysis of the lavage fluid from both lines of SP-C/Sftpc -/- mice. Induced SP-C expression was localized to alveolar type II cells by immunostaining with an antibody to proSP-C. Line 55.3 SP-C/Sftpc -/- mice were maintained on or off dox for 7 days and infected with 2.6x10⁷ RSV pfu. On day 3 post RSV infection total inflammatory cell counts were reduced in the lavage of dox treated 55.3 SP-C/Sftpc -/- mice (p = 0.004). The percentage of neutrophils was reduced (p = 0.05). The viral titers of lung homogenates from dox treated 55.3 SP-C/Sftpc -/- mice were decreased relative to 55.3 SP-C/Sftpc -/- mice without dox (p = 0.01). The cytokine response of Sftpc -/- type II cells to RSV was increased over that of Sftpc +/+ cells.

Conclusions

Transgenic restoration of SP-C reduced inflammation and improved viral clearance in the lungs of SP-C deficient mice. The loss of SP-C in alveolar type II cells compromises their response to infection. These findings show that the restoration of SP-C in Sftpc -/- mice in response to RSV infection is a useful model to determine parameters for therapeutic intervention.     

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

Key message

The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.

Abstract

Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.     

心脏特异表达Dhcr24转基因小鼠的建立和表型分析

目的建立心脏特异表达小鼠24-脱氢胆固醇还原酶基因（Dhcr24）转基因小鼠,研究该基因在心脏中表达对小鼠心脏发育,形态和功能维持中的作用。方法RT-PCR法克隆小鼠24-脱氢胆固醇还原酶基因,把Dhcr24基因插入-αMHC启动子下游,构建转基因表达载体,通过显微注射法建立Dhcr24 C57BL/6J转基因小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,RT-PCR和Western Blotting检测基因表达水平,光学显微镜和超声检测不同月龄Dhcr24转基因小鼠心脏的组织结构改变。结果建立了2个品系的心脏特异表达Dhcr24转基因小鼠。转入的Dhcr24基因在心脏组织的表达水平超过内源性Dhcr24的3倍。心脏组织学和超声检查证实：Dhcr24转基因小鼠的心室壁变厚,心腔变小,但心脏功能保持正常。结论成功建立了心脏特异表达Dhcr24转基因小鼠,Dhcr24基因在心脏组织的过度表达对小鼠心脏发育和功能维持中的作用需要进一步探讨。     

Chloroplast-targeted expression of the codon-optimized truncated cry1Ah gene in transgenic tobacco confers a high level of protection against insects

Key message

The study of insect-resistant transgenic tobacco provides a good foundation for the further application of the cry1Ah gene in other important crops.

Abstract

To improve transgene expression levels and insect resistance, the coding sequence of the novel Bacillus thuringiensis insecticidal gene cry1Ah (truncated cry1Ah) was modified according to the codon bias of the plant by increasing its GC content from the original 37 % to 48, 55, and 63 % (designated m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah, respectively). In addition, the m3-cry1Ah gene was linked with a transit peptide sequence for chloroplast-targeted expression (designated ctp-m3-cry1Ah). Four plant expression vectors were constructed harboring m1-cry1Ah, m2-cry1Ah, m3-cry1Ah, or ctp-m3-cry1Ah. A total of 23 transgenic tobacco lines were produced with the four constructs by Agrobacterium tumefaciens-mediated transformation. PCR, Southern hybridization, quantitative RT-PCR and ELISA indicated that the cry1Ah gene was not only integrated into the tobacco genome, but was also successfully expressed at the mRNA and protein levels. The Cry1Ah protein level in ctp-m3-cry1Ah plants reached 4.42 μg/g fresh weight, which was a 2- to 10-fold increase over the levels observed in m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah plants and resulted in the highest resistance to Helicoverpa armigera based on bioassays. Our results demonstrated that combining the codon optimization of cry1Ah gene with the targeting of Cry1Ah protein to the chloroplasts conferred a high level of protection against insects. The results of our experiments in tobacco, an important model system, provide a good foundation for enhancing the insecticidal efficacy of staple crops.     

Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis: IDENTIFICATION OF ALTERED METABOLIC PATHWAYS IN DHCR7 and SC5D DEFICIENCY*

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.Smith-Lemli-Opitz syndrome (SLOS¹; Online Mendelian Inheritance in Man 270400) is a multiple malformation syndrome with cognitive and behavioral deficiencies due to an inborn error of cholesterol synthesis. Typical findings in SLOS include dysmorphic facial features, limb defects, genital anomalies, growth retardation, cognitive disabilities, behavioral problems, and autistic features (for a review, see Ref. 1). The incidence of SLOS has been estimated to be on the order of 1/20,000–1/70,000 (1). SLOS is an autosomal recessive disorder caused by mutation of the 7-dehydrocholesterol reductase gene (DHCR7) (2–4). DHCR7 catalyzes the final step in the Kandutsch-Russel cholesterol biosynthetic pathway. Impaired DHCR7 activity results in increased 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (Fig. 1A). Lathosterolosis is a rare “SLOS-like” malformation syndrome due to mutations of lathosterol 5-desaturase (SC5D) (5–7). SC5D catalyzes the conversion of lathosterol to 7DHC. Thus, in lathosterolosis, like SLOS, there is a deficiency of cholesterol. However, the accumulating precursor sterol is lathosterol rather than 7DHC (Fig. 1A). Because of its rarity and the fact that all known cases of lathosterolosis were ascertained due to similarity with SLOS, the phenotypic spectrum of lathosterolosis has not been defined.Open in a separate windowFig. 1.Representative 2-DE maps of SLOS and lathosterolosis mouse brain proteins. A, SLOS and lathosterolosis are inborn errors of cholesterol synthesis. SLOS is caused by mutations in the DHCR7 gene. DHCR7 catalyzes the final step in cholesterol synthesis. Lathosterolosis is caused by mutations of the SC5D gene. Cholesterol levels are decreased in both SLOS and lathosterolosis, but the accumulating precursor sterol differs. In SLOS, 7DHC accumulates, whereas in lathosterolosis, the accumulating sterol is lathosterol. B, representative 2-DE maps of control (Dhcr7^+/+ and Sc5d^+/+), Dhcr7^{Δ3–5/Δ3–5}, and Sc5d^−/− mouse brain proteins. Eighty micrograms of the pooled protein sample from Dhcr7^+/+, Dhcr7^{Δ3–5/Δ3–5}, Sc5d^+/+, and Sc5d^−/− embryonic mouse brain tissues were separated on a pH 3–10 nonlinear IPG strip followed by electrophoretic separation on a 12% SDS-polyacrylamide gel. Acidic pH is to the left, and increased molecular mass is at the top. Compared with Dhcr7^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Dhcr7^{Δ3–5/Δ3–5} mouse brains are marked in Dhcr7^+/+ and Dhcr7^{Δ3–5/Δ3–5} mouse brain 2-DE maps, respectively. Compared with Sc5d^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Sc5d^−/− mouse brains are marked in Sc5d^+/+ and Sc5d^−/− mouse brain 2-DE maps, respectively. Supplemental Table 2 provides detailed information on the differentially expressed protein spots.Although the genetic and biochemical causes of SLOS are defined, the pathophysiological mechanisms contributing to specific malformations have not been delineated. The classic paradigm for the pathogenesis of an inborn error of metabolism includes the accumulation of a toxic precursor and/or deficiency of an essential product. In the case of SLOS, the observed defects are postulated to be caused, either singly or in combination, by cholesterol deficiency or the accumulation of 7DHC (8, 9).Cholesterol is an essential lipid with multiple critical functions. In addition to being a structural lipid in membranes and myelin, cholesterol is the precursor for bile acid, steroid hormone, neuroactive steroid, and oxysterol synthesis. In cellular membranes, cholesterol rafts are microdomains that function in receptor-mediated signal transduction. Functional defects in IgE receptor-mediated mast cell degranulation and cytokine production (10), N-methyl-d-aspartate receptor function (11), and serotonin 1A receptor ligand binding (12, 13) have been reported in SLOS. The altered sterol composition in SLOS affects the physiochemical properties and function of lipid rafts. Membrane domains incorporating 7DHC differ from those containing only cholesterol in protein composition (14), packing (15), and stability (16–18). Substitution of 7DHC for cholesterol also decreases membrane bending rigidity (19). In addition, model membranes mimicking SLOS membranes have been reported to exhibit atypical membrane organization (20) and curvature (19). These alterations may have functional consequences. Depletion of cholesterol from hippocampal membranes and replenishment with 7-dehydrocholesterol does not restore ligand binding activity of the serotonin 1A receptor despite the recovery of the overall membrane order (12). Cholesterol is also necessary for maturation and function of the hedgehog family of morphogens during embryonic development, and several mechanisms by which sonic hedgehog signaling might be impaired in SLOS have been proposed (21–23).To understand the pathophysiological processes underlying cognitive defects found in SLOS, we need to consider the potential detrimental effects of decreased cholesterol/functional sterol levels versus the potential toxic effects of increased 7DHC. To give insight into pathological effects due to cholesterol deficiency and precursor accumulation, we have produced mouse models deficient in either 7-dehydrocholesterol reductase (11) or lathosterol reductase (6) activity (Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/−, respectively). Although the two models are similar in many respects, significant differences exist. Dhcr7 pups have relatively few physical malformations other than a low frequency of cleft palate but die during the 1st day of life due to failure to feed (11). In contrast Sc5d mutant embryos are stillborn and have multiple developmental malformations (6). In addition, although secretory granule formation is altered in both models, consistent with differing physiochemical properties of the two precursor sterols, the specific changes differ between the two models (19). For these reasons, a comparison of the two models will provide insight into common mechanisms that are likely due to cholesterol/sterol deficiency and syndrome-specific mechanisms that are due to specific effects of one of the two precursors.We now report the use of two-dimensional electrophoresis (2-DE) mass spectrometry proteomics analysis to identify proteins with altered expression in brain tissue from both Dhcr7 and Sc5d mutants with the goal of identifying novel pathophysiological mechanisms contributing to the neurological deficits in these two inborn errors of cholesterol synthesis. Because our focus was on identifying processes that could contribute to abnormal neurological development, our analysis was focused on brain tissue from E18.5 embryos. This embryonic age was selected because the biochemical defect increases with embryonic age (6, 11), and it is the latest time point for which we could obtain viable tissue for both mutants. Western blot analysis was used to validate selected individual proteins and pathways. Functional annotation suggested that alterations in mevalonate metabolism, glycolysis, oxidative stress, apoptosis, protein biosynthesis, intracellular trafficking, and cytoskeleton may contribute to the pathology of inborn errors of cholesterol synthesis. In addition, our data are consistent with the hypothesis that both cholesterol deficiency and increased precursor sterol levels contribute to SLOS and lathosterolosis pathology.     

Expression profiling in APP23 mouse brain: inhibition of Aβ amyloidosis and inflammation in response to LXR agonist treatment

Background

Recent studies demonstrate that in addition to its modulatory effect on APP processing, in vivo application of Liver X Receptor agonist T0901317 (T0) to APP transgenic and non-transgenic mice decreases the level of Aβ_42. Moreover, in young Tg2576 mice T0 completely reversed contextual memory deficits. Compared to other tissues, the regulatory functions of LXRs in brain remain largely unexplored and our knowledge so far is limited to the cholesterol transporters and apoE. In this study we applied T0 to APP23 mice for various times and examined gene and protein expression. We also performed a series of experiments with primary brain cells derived from wild type and LXR knockout mice subjected to various LXR agonist treatments and inflammatory stimuli.

Results

We demonstrate an upregulation of genes related to lipid metabolism/transport, metabolism of xenobiotics and detoxification. Downregulated genes are involved in immune response and inflammation, cell death and apoptosis. Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Aβ. In primary LXR^wt but not in LXRα^-/-β^-/- microglia and astrocytes LXR agonists suppressed the inflammatory response induced by LPS or fibrillar Aβ.

Conclusion

The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation. An increased understanding of the LXR controlled regulation of Aβ aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.     

Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice

Background

Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth.

Principal Findings

By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus.

Significance

In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.     

Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice

Transgenic animals have been established for studying gene function, improving animals’ production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken β-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.