CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells.

Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3'-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.

Inducible knockdown of procollagen I protects mice from liver fibrosis and leads to dysregulated matrix genes and attenuated inflammation.

Organ fibrosis is characterized by a chronic wound-healing response, with excess deposition of extracellular matrix components. Here, collagen type I represents the most abundant scar component and a primary target for antifibrotic therapies. Liver fibrosis can progress to cirrhosis and primary liver cancer, which are the major causes of liver related morbidity and mortality. However, a (pro-)collagen type I specific therapy remains difficult and its therapeutic abrogation may incur unwanted side effects. We therefore designed tetracycline-regulated procollagen alpha1(I) short hairpin (sh)RNA expressing mice that permit a highly efficient inducible knockdown of the procollagen alpha1(I) gene in activated (myo-)fibroblasts, to study the effect of induced procollagen type I deficiency. Transgenic mice were generated using recombinase-mediated integration in embryonic stem cells or zinc-finger nuclease-aided genomic targeting combined with miR30-shRNA technology. Liver fibrosis was induced in transgenic mice by carbon tetrachloride, either without or with doxycycline supplementation. Doxycycline treated mice showed an 80-90% suppression of procollagen alpha1(I) transcription and a 40-50% reduction in hepatic collagen accumulation. Procollagen alpha1(I) knockdown also downregulated procollagens type III, IV and VI and other fibrosis related parameters. Moreover, this was associated with an attenuation of chronic inflammation, suggesting that collagen type I serves not only as major scar component, but also as modulator of other collagens and promoter of chronic inflammation.

mTOR inhibitor reverses autistic-like social deficit behaviours in adult rats with both Tsc2 haploinsufficiency and developmental status epilepticus.

Epilepsy is a major risk factor for autism spectrum disorder (ASD) and complicates clinical manifestations and management of ASD significantly. Tuberous sclerosis complex (TSC), caused by TSC1 or TSC2 mutations, is one of the medical conditions most commonly associated with ASD and has become an important model to examine molecular pathways associated with ASD. Previous research showed reversal of autism-like social deficits in Tsc1 +/- and Tsc2 +/- mouse models by mammalian target of rapamycin (mTOR) inhibitors. However, at least 70 % of individuals with TSC also have epilepsy, known to complicate the severity and treatment responsiveness of the behavioural phenotype. No previous study has examined the impact of seizures on neurocognitive reversal by mTOR inhibitors. Adult Tsc2 +/- (Eker)-rats express social deficits similar to Tsc2 +/- mice, with additive social deficits from developmental status epilepticus (DSE). DSE was induced by intraperitoneal injection with kainic acid at post-natal days P7 and P14 (n = 12). The experimental group that modelled TSC pathology carried the Tsc2 +/- (Eker)-mutation and was challenged with DSE. The wild-type controls had not received DSE (n = 10). Four-month-old animals were analysed for social behaviour (T1), then treated three times during 1 week with 1 mg/kg everolimus and finally retested in the post-treatment behavioural analysis (T2). In the experimental group, both social interaction and social cognition were impaired at T1. After treatment at T2, behaviour in the experimental group was indistinguishable from controls. The mTOR inhibitor, everolimus, reversed social deficit behaviours in the Tsc2 haploinsufficiency plus DSE animal model to control levels.

A Limited Role for the Cell Cycle Regulator Cyclin A1 in Murine Leukemogenesis.

The quest for novel therapeutic targets in acute myeloid leukemia (AML) is still ongoing. One of such targets, cyclin A1, was shown to be overexpressed in AML including AML stem cells. However, the function of cyclin A1 in AML is largely unknown, and the data on its impact on patients' survival remain controversial. Therefore, we developed a transgenic mouse model of stem cell-directed inducible cyclin A1 overexpression and crossed these mice with PML-RARα-knockin mice, which develop an AML M3-like phenotype. To observe the effects of cyclin A1 loss-of-function, we also crossed PML-RARα-knockin mice to cyclin A1-knockout mice. Neither overexpression nor loss of cyclin A1 significantly altered leukemogenesis in PML-RARα-knockin mice. These findings imply that upregulation of cyclin A1 is not essential for leukemogenesis. Our data suggest that cyclin A1 does not represent a suitable target for AML therapy.

γ-Aminobutyric A receptor (GABA(A)R) regulates aquaporin 4 expression in the subependymal zone: relevance to neural precursors and water exchange.

Activation of γ-aminobutyric A receptors (GABA(A)Rs) in the subependymal zone (SEZ) induces hyperpolarization and osmotic swelling in precursors, thereby promoting surface expression of the epidermal growth factor receptor (EGFR) and cell cycle entry. However, the mechanisms underlying the GABAergic modulation of cell swelling are unclear. Here, we show that GABA(A)Rs colocalize with the water channel aquaporin (AQP) 4 in prominin-1 immunopositive (P(+)) precursors in the postnatal SEZ, which include neural stem cells. GABA(A)R signaling promotes AQP4 expression by decreasing serine phosphorylation associated with the water channel. The modulation of AQP4 expression by GABA(A)R signaling is key to its effect on cell swelling and EGFR expression. In addition, GABA(A)R function also affects the ability of neural precursors to swell in response to an osmotic challenge in vitro and in vivo. Thus, the regulation of AQP4 by GABA(A)Rs is involved in controlling activation of neural stem cells and water exchange dynamics in the SEZ.

Postnatal subventricular zone progenitors switch their fate to generate neurons with distinct synaptic input patterns.

New granule cell neurons (GCs) generated in the neonatal and adult subventricular zone (SVZ) have distinct patterns of input synapses in their dendritic domains. These synaptic input patterns determine the computations that the neurons eventually perform in the olfactory bulb. We observed that GCs generated earlier in postnatal life had acquired an 'adult' synaptic development only in one dendritic domain, and only later-born GCs showed an 'adult' synaptic development in both dendritic domains. It is unknown to what extent the distinct synaptic input patterns are already determined in SVZ progenitors and/or by the brain circuit into which neurons integrate. To distinguish these possibilities, we heterochronically transplanted retrovirally labeled SVZ progenitor cells. Once these transplanted progenitors, which mainly expressed Mash1, had differentiated into GCs, their glutamatergic input synapses were visualized by genetic tags. We observed that GCs derived from neonatal progenitors differentiating in the adult maintained their characteristic neonatal synapse densities. Grafting of adult SVZ progenitors to the neonate had a different outcome. These GCs formed synaptic densities that corresponded to neither adult nor neonatal patterns in two dendritic domains. In summary, progenitors in the neonatal and adult brain generate distinct GC populations and switch their fate to generate neurons with specific synaptic input patterns. Once they switch, adult progenitors require specific properties of the circuit to maintain their characteristic synaptic input patterns. Such determination of synaptic input patterns already at the progenitor-cell level may be exploited for brain repair to engineer neurons with defined wiring patterns.

Global increase of p16INK4a in APC-deficient mouse liver drives clonal growth of p16INK4a-negative tumors.

UNLABELLED: Reduction of ß-catenin (CTNNB1) destroying complex components, for example, adenomatous polyposis coli (APC), induces ß-catenin signaling and subsequently triggers activation of genes involved in proliferation and tumorigenesis. Though diminished expression of APC has organ-specific and threshold-dependent influence on the development of liver tumors in mice, the molecular basis is poorly understood. Therefore, a detailed investigation was conducted to determine the underlying mechanism in the development of liver tumors under reduced APC levels. Mouse liver at different developmental stages was analyzed in terms of ß-catenin target genes including Cyp2e1, Glul, and Ihh using real-time RT-PCR, reporter gene assays, and immunohistologic methods with consideration of liver zonation. Data from human livers with mutations in APC derived from patients with familial adenomatous polyposis (FAP) were also included. Hepatocyte senescence was investigated by determining p16(INK4a) expression level, presence of senescence-associated ß-galactosidase activity, and assessing ploidy. A ß-catenin activation of hepatocytes does not always result in ß-catenin positive but unexpectedly also in mixed and ß-catenin-negative tumors. In summary, a senescence-inducing program was found in hepatocytes with increased ß-catenin levels and a positive selection of hepatocytes lacking p16(INK4a), by epigenetic silencing, drives the development of liver tumors in mice with reduced APC expression (Apc(580S) mice). The lack of p16(INK4a) was also detected in liver tumors of mice with triggers other than APC reduction. IMPLICATIONS: Epigenetic silencing of p16(Ink4a) in selected liver cells bypassing senescence is a general principle for development of liver tumors with ß-catenin involvement in mice independent of the initial stimulus.

Transforming growth factor beta receptor 2 (TGFBR2) changes sialylation in the microsatellite unstable (MSI) Colorectal cancer cell line HCT116.

Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.

Construction and application of an inducible system for homogenous expression levels in bulk cell lines.

Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a beta-catenin-ERalpha fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-beta-catenin-ERalpha vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.

Highly B lymphocyte-specific tamoxifen inducible transgene expression of CreER T2 by using the LC-1 locus BAC vector.

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio-temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte-specific tamoxifen-inducible Cre transgenic mouse strain, LC-1-hCD19-CreER(T2). We utilized the human CD19 promoter for expression of the tamoxifen-inducible Cre recombinase (CreER(T2)) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross-breeding the LC-1-hCD19-CreER(T2) strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC-1-hCD19-CreER(T2) strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse.

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.

Generation of inducible hepatitis C virus transgenic mouse lines.

Hepatitis C virus (HCV) is the causative agent of most cases of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) affecting more than 170 million people world-wide. Progress in elucidating the nature of HCV and the development of new therapeutic strategies is hampered fundamentally by the absence of adequate small animal models simulating natural HCV infection. The creation of conditional mouse lines with the tetracycline-controlled gene expression system holds new perspectives for simulation of wild-type HCV infection in a small animal model. Transgenic mice were established with tetracycline-inducible coexpression of HCV core or HCV open reading frame (ORF) and luciferase. In long-term induction experiments, mice were examined for immunopathological changes after expression of HCV proteins. Inducible and liver-specific expression of transgenes was detected by Western blot, immunoprecipitation, luciferase assay and in vivo imaging of bioluminescence of luciferase in genetically modified mice. Ectopic expression levels were determined quantitatively in the liver, kidney, heart and spleen of mice in the induced and non-induced state. During long-term induction an elevation of aminotransaminases (ALT) was observed only in HCV core/ORF-expressing mice, but HCV-specific immune response was not confirmed by in vitro immunological assays. The histology of liver sections provided evidence of steatosis, which was correlated with an inflammatory response. The inducible HCV-transgenic mouse lines provide further evidence of liver pathogenesis in the presence of inflammation during liver-specific expression of HCV proteins and offer new insights into the effects of temporally and spatially controlled protein expression of HCV.

Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo.

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTA(CMV) (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTA(CMV) transgenes, FerH expression was increased 6.0+/-1.1-fold (mean+/-S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2(S)-S2(LAP) (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2+/-4.8-fold (mean+/-S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3+/-0.9-fold (mean+/-S.D.), concurrent with a 4.5+/-2.1-fold (mean+/-S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.

Chronic hepcidin induction causes hyposideremia and alters the pattern of cellular iron accumulation in hemochromatotic mice.

We report the generation of a tetracycline-regulated (Tet ON) transgenic mouse model for acute and chronic expression of the iron regulatory peptide hepcidin in the liver. We demonstrate that short-term and long-term tetracycline-dependent activation of hepcidin in adult mice leads to hypoferremia and iron-limited erythropoiesis, respectively. This clearly establishes the key role of hepcidin in regulating the extracellular iron concentration. We previously demonstrated that, when expressed early in fetal development, constitutive transgenic hepcidin expression prevented iron accumulation in an Hfe-/- mouse model of hemochromatosis. We now explore the effect of chronic hepcidin expression in adult Hfe-/- mice that have already developed liver iron overload. We demonstrate that induction of chronic hepcidin expression in 2-month-old Hfe-/- mice alters their pattern of cellular iron accumulation, leading to increased iron in tissue macrophages and duodenal cells but less iron in hepatocytes. These hepcidin-induced changes in the pattern of cellular iron accumulation are associated with decreased expression of the iron exporter ferroportin in macrophages but no detectable alteration of ferroportin expression in the hepatocytes. We speculate that this change in iron homeostasis could offer a therapeutic advantage by protecting against damage to parenchymal cells.

Use of the tetracycline system for inducible protein synthesis in the kidney.

The great advantage of the tetracycline-inducible system lies in its ability to address a large variety of biological questions in a time-dependent and tissue-specific manner. This study describes a transgenic mouse line, rTA(LAP)-1, which produces the reverse tetracycline transactivator under control of the liver activator protein (LAP) promoter. Two reporter lines with luciferase and LacZ reporter genes were used to demonstrate predominant expression in the kidney and liver when doxycycline was added to the drinking water. In the kidney, transgene expression was found primarily in cortical proximal tubules. No luciferase and beta-galactosidase activity was detected in mice without doxycycline in the drinking water, which attests to the tight control of this system. One of the advantages of the tet system lies in its reversibility, and indeed, a virtually complete remission of transgene activity in both the kidney and liver was observed when doxycycline was withdrawn. Also examined was transactivator activity during development by exposing the mothers producing the reverse transactivator to doxycycline before mating. Transgene activity was detected in newborn kidneys and liver, indicating that sufficient amounts of doxycycline had crossed the placental barrier. During nephron development, the LAP promoter appeared to be only active in the more mature proximal tubules. Finally, the rTA(LAP)-1 line was used to inducibly express the human PKD2 cDNA in proximal tubules of transgenic mice, but no cystic changes were detected, even after 6 mo of induction.