Cre-mediated DNA Recombination Research Articles

Establishment and characterization of a novel reverse genetic system of BK polyomavirus.

BK polyomavirus (BKV) is a small non-enveloped DNA virus. BKV infection or reactivation may cause BKV-associated nephropathy and hemorrhagic cystitis in immunosuppressed transplant recipients. No effective antivirals or prevention strategies are available against BKV infections. The current BKV reverse system employs the transfection of purified full-length linear viral genomes released by enzyme digestion from BKV genomic plasmids. The method is laborious and often results in variable DNA yield and quality, which can affect the efficiency of transfection and subsequent formation of circular viral genomes in cells. In this study, we report the generation of circular viral genomes by Cre-mediated DNA recombination in cells directly transfected with BKV precursor genomic plasmids. The novel system supported efficient viral expression and replication, and produced a higher level of infectious virions compared with the transfection with linear BKV genomes. Furthermore, we successfully constructed recombinant BKV capable of reporter gene expression. In conclusion, the novel BKV reverse genetic system allows for simpler manipulation of BKV genome with better virus yield, providing a tool for the study of BKV life cycle and antiviral screening.

Extracellular communication between brain cells through functional transfer of Cre mRNA mediated by extracellular vesicles

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo over time. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected invivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain, exhibiting an increment of more than 10-fold over 4months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEV transfer at physiological levels, which will shed light on the role of bdEVs in neural communication within the brain and beyond.

Identification of novel B-1 transitional progenitors by B-1 lymphocyte fate-mapping transgenic mouse model Bhlhe41 dTomato-Cre.

B-1 lymphocytes exhibit specialized roles in host defense against multiple pathogens. Despite the fact that CD19+CD93+B220lo/- B cells have been identified as B-1 progenitors, the definition for B-1 progenitors remains to be elucidated as CD19+CD93+B220+ B cells are capable to give rise to B-1 cells. Given that transcription factor Bhlhe41 is highly and preferentially expressed in B-1 cells and regulates B-1a cell development, we generated a transgenic mouse model, Bhlhe41dTomato-Cre , for fate mapping and functional analysis of B-1 cells. Bhlhe41dTomato-Cre mice efficiently traced Bhlhe41 expression, which was mainly restricted to B-1 cells in B-cell lineage. We showed an efficient and specific Cre-mediated DNA recombination in adult B-1 cells and neonatal B-1 progenitors rather than B-2 cells by flow cytometric analysis of Bhlhe41 dTomato-Cre/+ Rosa26 EYFP mice. Treatment of Bhlhe41 dTomato-Cre/+ Rosa26 iDTR mice with diphtheria toxin revealed a robust efficacy of B-1 cell depletion. Interestingly, using Bhlhe41 dTomato-Cre mice, we demonstrated that neonatal B-1 progenitors (CD19+CD93+B220lo/-) expressed Bhlhe41 and were identical to well-defined transitional B-1a progenitors (CD19+CD93+B220lo/-CD5+), which only gave rise to peritoneal B-1a cells. Moreover, we identified a novel population of neonatal splenic CD19hidTomato+B220hiCD43loCD5lo B cells, which differentiated to peritoneal B-1a and B-1b cells. Bhlhe41 deficiency impaired the balance between CD19hidTomato+B220lo/-CD5hi and CD19hidTomato+B220hiCD5lo cells. Hence, we identified neonatal CD19hidTomato+B220hiCD43loCD5lo B cells as novel transitional B-1 progenitors. Bhlhe41 dTomato-Cre/+ mouse can be used for fate mapping and functional studies of B-1 cells in host-immune responses.

A novel mouse line with epididymal initial segment-specific expression of Cre recombinase driven by the endogenous Lcn9 promoter.

Spermatozoa released from testes undergo a maturation process and acquire the capacity to fertilize ova through epididymal transit. The epididymis is divided into four regions, each with unique morphology, gene profile, luminal microenvironment and distinct function. To study the functions of relevant genes in the epididymal initial segment (IS), a novel IS-specific mouse model, Lcn9-Cre knock-in (KI) mouse line was generated via CRISPR/Cas9 technology. The TAG stop codon was replaced by a 2A-NLS-Cre cassette, resulting in the co-expression of Lcn9 and Cre recombinase. IS-specific Cre expression was first observed from postnatal day 17. Using the Rosa26tdTomato reporter mice, the Cre-mediated DNA recombination was detected exclusively in principal cells. The epididymal IS-specific Cre activity in vivo was further confirmed using Lcn9-Cre mice crossed with a mouse strain carrying Tsc1 floxed alleles (Tsc1flox/+). Cre expression did not affect either normal development or male fecundity. Different from any epididymis-specific Cre mice reported previously, the novel Lcn9-Cre mouse line can be used to introduce entire IS-specific conditional gene editing for gene functional study.

Calorie Restriction Increases the Number of Competing Stem Cells and Decreases Mutation Retention in the Intestine

SummaryCalorie restriction (CR) extends lifespan through several intracellular mechanisms, including increased DNA repair, leading to fewer DNA mutations that cause age-related pathologies. However, it remains unknown how CR acts on mutation retention at the tissue level. Here, we use Cre-mediated DNA recombination of the confetti reporter as proxy for neutral mutations and follow these mutations by intravital microscopy to identify how CR affects retention of mutations in the intestine. We find that CR leads to increased numbers of functional Lgr5+ stem cells that compete for niche occupancy, resulting in slower but stronger stem cell competition. Consequently, stem cells carrying neutral or Apc mutations encounter more wild-type competitors, thus increasing the chance that they get displaced from the niche to get lost over time. Thus, our data show that CR not only affects the acquisition of mutations but also leads to lower retention of mutations in the intestine.

Injection-Based Delivery of Cell-Permeable Peptide-Tagged Cre.

The technique of Cre-mediated DNA recombination at loxP sites has been used widely in manipulation of the genome in cultured cells and in living organisms. Local delivery of Cre recombinase protein tagged with a cell-penetrating (or permeable) peptide (Cre-CPP) has the advantage of additional spatial and temporal control when compared to genetic delivery methods. In this chapter, we describe protocols for injection-based intramuscular delivery of Cre-CPP dissolved in hydrogel to skeletal muscle and by ultrasound-guided injection to cardiac muscle in mice.

Single-Cell Lineage Analysis of Oogenesis in Mice.

Lineage analysis is widely used because it provides a very powerful tool for characterizing the developmental behavior of the cells in vivo. In this chapter, we describe a particularly informative variant of lineage analysis that we term "single-cell lineage analysis". As in traditional lineage analysis, the method employs a Tamoxifen (Tmx)-inducible CAGCreER mouse line, which is crossed to an R26R reporter line that can be activated by Cre-mediated DNA recombination. However, instead of driving CreER at a high level within a subset of cells defined by a particular promoter, CreER is driven with a generic promoter that is active in essentially all cells throughout the lifespan of the mouse. Specificity comes from using only a very low dose of Tmx so that just a few random, widely separated individual cells undergo recombination and become labeled. The growth and behavior of most such initially marked cells can subsequently be followed over time because each one forms a growing clone of marked cells that does not overlap with other clones due to their rarity. Following individual cell growth patterns provides much more information than can be derived from traditional lineage analysis, which relies on promoter specificity and uses high doses of Tmx that cannot resolve the behavior of single cells. We illustrate the value of single-cell lineage analysis using a recent study of fetal germ cell development and a recent search for female germ-line stem cells in adult mouse ovaries.

Abstract 3095: Notch-induced choroid plexus tumor arises from epithelial progenitor and depends on sonic hedgehog signaling for growth

Abstract Introduction: Choroid plexus tumors are rare intraventricular papillary neoplasms arising from choroid plexus (CP) epithelium. They occur most often in childhood and comprise 10-20% of all brain tumors in infants. Choroid plexus papilloma is a benign tumor, whereas choroid plexus carcinoma is malignant and most commonly found in pediatric population. Knowledge of the oncogenic processes in CP tumor formation will profoundly impact the development of new and molecular targeted therapies. Previous studies implicate Notch signaling in CP tumorigenesis. However, mechanisms underlying Notch-driven CP tumor formation remain unclear. Animal models of CP tumors have great potential to facilitate the discovery and development of molecular diagnostics and targeted therapies for these rare diseases. Methods and Results: We used the Rosa26-NICD1 strain which will constitutively express the intracellular domain of Notch 1 (NICD1) from the Rosa26 locus after Cre-mediated DNA recombination. The hindbrain CP targeted for NICD1 expression was significantly enlarged with well circumscribed, globular, cauliflower like masses. Histological analysis revealed that CP from NICD1-expressing mutant mice contained an abnormal growth in the 4th ventricle consistent with properties of choroid plexus tumor. Tumor cells exhibit enhanced proliferation transiently after birth: tumor cell proliferation was highest at birth, then gradually decreased and completely stopped at 2 weeks after birth. While CP tumor cells expressed early CP lineage markers such as Lmx1a and Otx2, they didn’t express Aquaporin 1 (AQP1), marker for differentiated CP epithelial cells, suggesting lack of terminal differentiation. We further showed that CP tumor arises during development as ectopic Lmx1a+ cells in CP and exhibit properties similar to those of CP epithelium progenitors. However, unlike progenitor cells, tumor cells fail to exit the cell cycle and differentiate into mature epithelial cells. We characterized changes in gene expression in tumor cells by quantitative RT-PCR. Surprisingly, we detected increased levels of Shh, Gli1 and N-Myc, effectors of the Sonic Hedgehog (Shh) pathway, in proliferating tumor cells. CP tumor cells cultured in serum-free conditions underwent rapid proliferation in the presence of Shh. Ultra-structural studies revealed lack of multi-ciliation differentiation in tumors cells, whereas the expression of Foxj1 and Midas, crucial regulators of cilia differentiation, was significantly downregulated. Conclusions: We developed a genetic model of CP tumor driven by Notch signaling activation. Notch 1-induced CP tumor arises during development from CP epithelial progenitors but lack terminal differentiation and multi-ciliation of mature CP epithelial cells. The proliferation of Notch 1-induced tumors is supported by Shh pathway, indicating Shh pathway as a therapeutic target for Notch-driven CP tumors. Citation Format: Haotian Zhao, Li Li, Katie Picotte. Notch-induced choroid plexus tumor arises from epithelial progenitor and depends on sonic hedgehog signaling for growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2014-3095

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R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation

The aim of this study was to investigate whether direct injection of nonviral DNA into the oviductal lumen and subsequent in vivo electroporation leads to in vivo gene transfer in mouse preimplantation embryos present within an oviduct, as an alternative to the pre-existing pronuclear microinjection-based transgenesis. With this technique, effects of expression of the gene of interest (GOI) on mouse preimplantation development can be monitored with relative ease. Superovulated 4-week-old B6C3F1 female mice (hybrids between C57BL/6N and C3H/HeN) were mated with adult B6C3F1 male mice. Two days later, females that had been identified as pregnant, based on the presence of copulation plugs, were injected with 1 µl of a solution containing an enhanced green fluorescent protein (EGFP) expression plasmid (0.5 µg) and 0.05% trypan blue. The entire oviduct was then electroporated using tweezer-type electrodes with 8 square-wave pulses of 50 V each with 50-ms duration. The next day, the 8-cell stage embryos were collected, and their number, morphology, and EGFP-derived fluorescence were recorded. Of the 12 oviducts (6 females used) examined, 3 contained fluorescent 8-cell stage embryos (33%, 19/58 tested), but the intensity of fluorescence varied among the embryos. In total, 10% (19/192 tested) of the embryos were fluorescent and the fluorescence was maintained in these embryos after 1 day of culture. However, the fluorescence disappeared in the late gestational stage fetuses, and the transgenes could not be detected. Our results indicate that it is possible to transfect in vivo preimplantation embryos, although the success rate appears to be relatively low and gene expression is transient. This technology may provide a new method for manipulating preimplantation embryos in vivo, by using, for example, Cre-mediated conditional DNA recombination.

Conditional gene expression in hepatitis C virus transgenic mice without induction of severe liver injury using a non-inflammatory Cre-expressing adenovirus

We previously established inducible-hepatitis C virus (HCV) transgenic mice, which expressed the HCV gene (nucleotides 294–3435) encoding the core, E1, E2, and NS2 proteins. The expression of these proteins is regulated by the Cre/loxP system and an adenovirus vector (AdV) that expresses Cre DNA recombinase (Cre) controlled by the CAG promoter (AxCANCre). Recent studies have demonstrated that AxCANCre injection alone results in severe liver injury by induction of the adenovirus protein IX (Ad-pIX) gene. As a result, HCV protein expression in transgenic mice livers was only short-term. In contrast, the EF1α promoter-bearing AdV induces slight Ad-pIX gene expression without inducing severe liver injury. Therefore, in the present study, we developed a Cre-expressing AdV that bears the EF1α promoter (AxEFCre) to express HCV protein in the transgenic mouse livers. In the non-transgenic mice injected with AxCANCre, alanine aminotransferase (ALT) levels were elevated and severe liver inflammation occurred; this was not observed in AxEFCre-injected mice. In contrast, AxEFCre-injected HCV transgenic mice showed milder liver inflammatory responses that were clearly due to HCV protein expression. Moreover, the AxEFCre injection enabled the transgenic mice to persistently express HCV protein. These results indicate that use of AxEFCre efficiently promotes Cre-mediated DNA recombination in vivo without a severe hepatitis response to AdV. This inducible-HCV transgenic mouse model using AxEFCre should be useful for research on HCV pathogenesis.

Natural killer cells target HCV core proteins during the innate immune response in HCV transgenic mice

The mechanism of the innate immune response to hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We used HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins regulated by the Cre/loxP switching expression system, to examine the innate immune response to HCV structural proteins. Twelve hours after HCV transgene expression, HCV core protein levels in Tg mouse livers were 15-47 pg/mg. In contrast, in Tg mice with a depletion of natural killer (NK) cells, we observed much higher levels of HCV core proteins (1,597 pg/ml). Cre-mediated genomic DNA recombination efficiency in the HCV-Tg mice was strongly observed in NK cell-depleted mice between 0.5 and 1 day as compared to non-treated mice. These data indicated that NK cells participate in the elimination of core-expressing hepatocytes in the innate immune responses during the acute phase of HCV infection.

S1684 Sprouty-2 Enhances Cell Proliferation and Induces Resistance to Apoptosis Through IGF-1R Pathway in Colon Cancer Cells

Background: The Caudal-related homeobox (Cdx) transcription factor Cdx2 is a homeodomain protein related to the Drosophila Caudal gene. It is an important regulator of intestinespecific gene expression and columnar morphogenesis. It plays a key role in intestinal cell differentiation, cell to cell adhesion, columnar morphogenesis, and proliferation. Cdx2 has been implicated in a number of human disease processes including colon cancer and intestinal metaplasia. The purpose of this study is to develop a Cre-regulated Cdx2 expression model to facilitate the study of these complex and multi-staged processes. Other inducible systems generally yield a leaky expression that can blunt the Cdx2 response. A Cre-inducible system would not share this limitation. Methods: We have subcloned a Flag-Cdx2 cDNA into a Cre-regulated expression vector. In this construct, Cdx2 is linked in frame to a histone 2BRFP(Cherry) reporter. In between is a picornavirus 2A peptide directing cotranslational cleavage of the linked proteins, thereby releasing the Flag-Cdx2. Upstream to this sequence is a _eta_actin promoter sequence (with CMV enhancer sequence) to drive expression of the bicistronic mRNA. In between these is a loxP flanked cassette that includes a PGK-neo selectable marker and a SV40 virus tpA transcriptional stop sequence. Once integrated, this locus will remain silent until expression of Cre recombinase. This expression vector is cotransfected along with an expression vector for CreER(T2). Subsequent use of Tamoxifen enables drug-dependent control of Cre-mediated DNA recombination. Results: The Creresponsive Cdx2 clone was created and confirmed by DNA sequencing. 293T cells were transiently transfected with this construct, with and without a Cre expression vector. In the absence of Cre recombinase, there was immunofluorescent H2B detected. Conversely, in the presence of Cre co-transfection, reporter immunofluorescent product was strongly observed within cells. In addition, a whole cell lysate was prepared from these cells and analyzed by Western blot using anti-Cdx2 and anti-Flag-Cdx2 probes. Results showed that Flag-Cdx2 was identified exclusively in the Cdx2 clone + Cre co-transfection sample only. Stably transfected HCT116, DLD1, and Colo 205 cells are currently being raised for studies of the effects of Cdx2 induction on cell proliferation and cell to cell adhesion. Conclusions: We have developed a novel Cre-recombinase regulated Cdx2 expression system allowing for the spatiotemporal study of Cdx2 in both In Vitro and In Vivo models. Further studies with stable transfection of our Cdx2-inducible clone into colon cancer cell lines are underway.

Abstract 3969: Stromal depletion of transforming growth factor receptor 2 promotes the development of forestomach squamous cell carcinoma

Abstract Deletion or attenuation of transforming growth factor beta (TGFβ) signaling pathway has significant impact on human tumor development and progression. Specific deletion of transforming growth factor receptor 2 gene (Tgfbr2) in a subset of fibroblast (Tgfbr2fspKO, cre-mediated DNA recombination driven by fibroblast specific protein, FSP) specific promoter, leads to the development of squamous cell carcinoma of the forestomach in 100% of Tgfbr2fspKO mice by 6 Wks of Age. Here we dissect the underlying mechanisms. We first confirmed that deletion of Tgfbr2 is primarly restricted to the stromal components but not epithelial cells using TBR2 (TGFβR2) immunofluorescence and immunohistochemistry, which was done in both tumor tissue sections and cancer epithelial cells derived from tgfbr2fspKO mice. The functionality of TGFβ signaling was further examined in vivo by p-smad2 expression and nuclear localization which was clearly observed in the epithelial but not the stromal components. There was also a clear nuclear localization of p-smad2 protein in cultured cancer epithelial cells when treated with TGFβ1 (10 ng/ml). We further observed that stromal deletion of Tgfbr2 resulted in elevated proliferation of fibroblasts. In addition, there was a significantly increased inflammation in forestomach lesion which was featured by increased CD45+ leukocyte infiltration and elevated expression of inflammatory mediators COX2, NFkB, Akt. Interestingly, the alterations in epithelial cell compartment in Tgfbr2fspKO mice is very evident compared with floxed control littermates Tgfbr2fspKO mice, showing a significant enrichment of epithelial cancer stem cells. Our data demonstrate the importance of stromal TGFβ signaling mediated inflammation and carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3969.

Split-CreERT2: Temporal Control of DNA Recombination Mediated by Split-Cre Protein Fragment Complementation

BackgroundDNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2.Methodology/Principal FindingsIn the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC50-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM.Conclusions/SignificanceThese characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.

612 WEEK 8 HCV-RNA IS THE OPTIMAL PREDICTOR OF RELAPSE IN NON-RVR PATIENTS WITH GENOTYPE 1/4 RANDOMISED TO 48 OR 72 WEEKS PEG-IFN ALFA-2A PLUS RBV

The mechanism of innate immune response to the hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We examined the innate immune response to the HCV structural proteins using HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching expression system. The level of HCV core protein was 15-47 pg/mg of total protein in the HCV Tg mice livers at 12 h after the HCV transgene expression. In contrast, we found a high level of expression of HCV core protein (1597 pg/mL) in transgenic mice with a large depletion of natural killer (NK) cells. Cre-mediated genomic DNA recombination efficiency in the HCV Tg mice was was strongly observed in NK cell depleted mice, compared to non-treated mice between 0.5 and 1 day.These data indicated that in the early naive immune responses the NK cells have important roles in initial recognition of HCV protein and the later elimination of core expressed hepatocytes.

609 BASELINE CHARACTERISTICS AND WEEK-4 RESPONSE AMONG 1546 CHC PATIENTS INFECTED WITH HCV GENOTYPE 1, 2, 3 AND 4: INTERIM RESULTS OF THE PROPHESYS COHORT

The mechanism of innate immune response to the hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We examined the innate immune response to the HCV structural proteins using HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching expression system. The level of HCV core protein was 15-47 pg/mg of total protein in the HCV Tg mice livers at 12 h after the HCV transgene expression. In contrast, we found a high level of expression of HCV core protein (1597 pg/mL) in transgenic mice with a large depletion of natural killer (NK) cells. Cre-mediated genomic DNA recombination efficiency in the HCV Tg mice was was strongly observed in NK cell depleted mice, compared to non-treated mice between 0.5 and 1 day.These data indicated that in the early naive immune responses the NK cells have important roles in initial recognition of HCV protein and the later elimination of core expressed hepatocytes.

Noggin Suppression Enhances in Vitro Osteogenesis and Accelerates in Vivo Bone Formation

Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

Cre-estrogen receptor-mediated hepatitis C virus structural protein expression in mice

Hepatocyte apoptosis is an important feature of liver injury in hepatitis C virus (HCV) infection. However, the mechanism of apoptosis and consequences on disease progression in vivo have not been investigated fully in part due to the lack of adequate small animal models. In this study, transgenic (tg) mice were produced that express conditionally HCV structural proteins (core, E1, E2 and p7) in the liver following Cre-mediated DNA recombination. Using a novel Cre-estrogen receptor fusion protein (Cre-ER) induction strategy, tamoxifen was injected intraperitoneally (i.p.), which induced Cre nuclear translocation, transgene recombination and HCV protein expression in the liver. Hepatic expression of HCV core and envelope proteins resulted in increased hepatocyte apoptosis, detected by the TUNEL assay, between 7 and 33 days after induction. These results were confirmed by the presence of increased levels of apoptosis-associated cytokeratin 18 (CK-18) in the sera of the same animals. The presence of cleaved caspase-3 and elevated levels of CHOP/GADD153 in the liver suggests an endoplasmic reticulum (ER) stress-associated apoptosis mechanism. This study suggests an in vivo correlation between HCV structural protein expression, ER stress and hepatocyte apoptosis, implicating a potentially important mechanism of HCV pathogenesis.