56; 95% CI: 0.44–0.72). However, a higher rate of °III and °IV adverse events

was observed under systemic treatment (56% vs 6%). Similar results were obtained in the phase III ACTS-GC trial from Japan, evaluating the effect of adjuvant treatment with the oral 5-FU analogue S1 after D2-gastrectomy for GC [34]. The 5-year overall survival (OS) was 71.7% in patients receiving adjuvant treatment compared to 61.1% in the surgery-only group (HR 0.669; 95% CI: 0.540–0.828). A retrospective analysis of data from 10,251 patients in the US American SEER database evaluated the effect of pre- or post-surgery radiation in patients undergoing surgical gastrectomy for GC [35]. Concerning the entire cohort, there was no survival benefit for patients receiving any kind of radiation. Selective assessment of patients with positive lymph node involvement PF-562271 purchase revealed improved median overall and 5-year survival rates (pre-op. radiation: p = .0261; post-op. radiation: p < .001). However, retrieval of more than 15 lymph nodes during primary surgery was an independent predictor of survival in multivariate

analysis. As the outcome of patients with advanced GC is still poor, several this website regimens of systemic chemotherapy have been further assessed concerning their effectiveness in the palliative setting. In the Austrian GASTRIC-II trial, the combination of oxaliplatin, irinotecan, and cetuximab has been applied in 51 patients with advanced GC [36]. In 35 patients accessible for response evaluation, there was an overall response rate (ORR) of 23% with a median

time to progression (TTP) of 24.8 weeks and a median OS of 38.1 weeks for patients with wild-type status of the K-ras gene. Main toxicity was acceptable, however, with neutropenia in 35% (°III in nine and °IV in one patient, respectively), thrombocytopenia in 33%, anemia in 73%, peripheral polyneuropathy in 37%, nausea in 45%, diarrhea in 57%, and fatigue in 37% of patients. In a Chinese phase II trial, a modified XELIRI (capecitabine plus irinotecan; Xeloda®, Roche Pharmaceuticals, Basel, Switzerland) regimen was reported to be adequately safe and effective (ORR 43.7%) median TTP 5.6 months, MCE公司 OS 11.0 months)) [37]. Grade III/IV adverse events were neutropenia (15.6%), anorexia (9.4%), nausea (9.4%), vomiting (6.3%), and diarrhea (6.3%). Despite strong data from earlier studies supporting the addition of docetaxel to platinum/5-FU-based regimens, there are still phase II trials with conflicting results. A study from Turkey could not demonstrate a significant effect by addition of docetaxel on neither median OS (6.5 vs 8.7 months) or TTP (4.4 vs 65.2 months) in a cohort of 70 patients [38]. However, a Greek study demonstrated ORR of 59% and a median survival of 18.0 months for patients receiving docetaxel, oxaliplatin, and capecitabine with acceptable toxicity [39].

Magalhães et al. [6] found that H. pylori infection results in an increased expression of syndecan-4 when infections are sustained by CagA positive strains and provided in vitro evidence that CagA, but mainly Fostamatinib supplier CagE, is required to induce membrane-bound syndecan-4 expression. A global overview of the complexity of H. pylori OMPs gene expression was carried out by Odenbreit et al. [7] who studied the expression of eight OMPs (AlpA, AlpB, SabA, BabA, BabB, BabC, HP0227, OipA) in H. pylori isolates obtained

from the antrum of infected children. The hypothesis that, to adapt to a changing niche, H. pylori genome co-evolves with host response has been pursued by Giannakis et al. [8]. In a genome-wide analysis, H. pylori isolates obtained from the gastric corpus of multiple patients with variable gastric pathology the authors found that isolates differed selleck kinase inhibitor markedly between patients, but the H. pylori population within an individual was largely clonal and remained stable over a period of at least 4 years. By analyzing the transcriptome of infected gastric epithelial progenitors, the authors

identified Serpin-1, several protein tyrosine phosphatases and superoxide dismutase 2 among the highest upregulated genes, and Cdkn2c, a tumor suppressor gene, among the most strongly downregulated genes in H. pylori-infected patients with chronic atrophic gastritis and with gastric cancer, MCE but failed to find any new disease-associated gene. A genome-wide map of H. pylori transcriptional start sites (TSS) and operons was provided by Sharma et al. [9], which complemented genomic

sequence and global protein–protein interaction map of the H. pylori strain 26695. Uncoupling of polycistrons and genome-wide antisense transcription (27% of the primary TSS are also antisense TSS) contribute to the high complexity of H. pylori gene expression. Antisense TSS for 22/34 putative phase variable genes involved in LPS biosynthesis, surface structure and DNA restriction/modification were identified and this might well represent a new mechanism of controlling surface structure variations and host interactions. An increased proliferation not balanced by an increase in apoptosis has been postulated as a putative cause of H. pylori-associated gastric carcinogenesis. Yan et al. [10] demonstrated both in vitro and in an in vivo mouse model that a rodent adapted H. pylori cag-positive strain activates the epidermal growth factor receptor (EGFR) through the ADAM-17 mediated release of heparin-binding-EGF. EGFR activation in epithelial cells resulted in activation of Akt, decreased Bax expression and increased Bcl-2 expression, the downstream targets that promote an anti-apoptotic response in H. pylori-infected epithelial cells. The sonic hedgehog (Shh) expression in gastric adenocarcinoma samples from both mice and humans was induced by H. pylori in a time-dependent manner.

Magalhães et al. [6] found that H. pylori infection results in an increased expression of syndecan-4 when infections are sustained by CagA positive strains and provided in vitro evidence that CagA, but mainly buy Venetoclax CagE, is required to induce membrane-bound syndecan-4 expression. A global overview of the complexity of H. pylori OMPs gene expression was carried out by Odenbreit et al. [7] who studied the expression of eight OMPs (AlpA, AlpB, SabA, BabA, BabB, BabC, HP0227, OipA) in H. pylori isolates obtained

from the antrum of infected children. The hypothesis that, to adapt to a changing niche, H. pylori genome co-evolves with host response has been pursued by Giannakis et al. [8]. In a genome-wide analysis, H. pylori isolates obtained from the gastric corpus of multiple patients with variable gastric pathology the authors found that isolates differed MEK inhibitor markedly between patients, but the H. pylori population within an individual was largely clonal and remained stable over a period of at least 4 years. By analyzing the transcriptome of infected gastric epithelial progenitors, the authors

identified Serpin-1, several protein tyrosine phosphatases and superoxide dismutase 2 among the highest upregulated genes, and Cdkn2c, a tumor suppressor gene, among the most strongly downregulated genes in H. pylori-infected patients with chronic atrophic gastritis and with gastric cancer, MCE but failed to find any new disease-associated gene. A genome-wide map of H. pylori transcriptional start sites (TSS) and operons was provided by Sharma et al. [9], which complemented genomic

sequence and global protein–protein interaction map of the H. pylori strain 26695. Uncoupling of polycistrons and genome-wide antisense transcription (27% of the primary TSS are also antisense TSS) contribute to the high complexity of H. pylori gene expression. Antisense TSS for 22/34 putative phase variable genes involved in LPS biosynthesis, surface structure and DNA restriction/modification were identified and this might well represent a new mechanism of controlling surface structure variations and host interactions. An increased proliferation not balanced by an increase in apoptosis has been postulated as a putative cause of H. pylori-associated gastric carcinogenesis. Yan et al. [10] demonstrated both in vitro and in an in vivo mouse model that a rodent adapted H. pylori cag-positive strain activates the epidermal growth factor receptor (EGFR) through the ADAM-17 mediated release of heparin-binding-EGF. EGFR activation in epithelial cells resulted in activation of Akt, decreased Bax expression and increased Bcl-2 expression, the downstream targets that promote an anti-apoptotic response in H. pylori-infected epithelial cells. The sonic hedgehog (Shh) expression in gastric adenocarcinoma samples from both mice and humans was induced by H. pylori in a time-dependent manner.

The remaining 461 patients (264 men, 197 women; age range 38–93 years; mean age 68.2 ± 8.7 years) were enrolled in this study. They consisted of 107 (23.2%) elderly subjects, aged selleck chemicals 75 years or over, and 354 non-elderly subjects. According to the estimate released by the National Cancer Center, Japan, the number of liver cancer mortalities in Japanese persons aged over 75 years increased, whereas that of subjects under 75 years decreased between 2004 and 2008.16 Additionally, the incidence of liver cancer continually increased in Japanese persons over 75 years

until 2005, whereas the incidence in persons under 75 years reached its peak in 2003.17 Therefore, we divided subjects into two groups (those <75 years and those ≥75 years) to analyze and discuss the strategy of treatment for elderly HCC patients. The indication for RFA treatment was that HCC consisted of five or fewer nodules, with each nodule having a maximum diameter of 30 mm, or that HCC consisted of a single tumor, regardless of size, and that hepatic function was not Child–Pugh grade C. SCH772984 nmr Radiofrequency ablation treatment was applied to cases (n = 226) that were not considered

to be suitable for resection for the following reasons: (i) impairment of liver function, and (ii) an excessive number of tumors or cardiopulmonary dysfunction. In addition, we applied RFA in cases (n = 235) where patients chose ablation therapy

even though surgery was also feasible. Exclusion criteria for RFA were: (i) 上海皓元医药股份有限公司 total bilirubin concentration over 3 mg/dL; (ii) platelet count under 30 000/mm3; (iii) prothrombin activity under 50%; (iv) ascites that could not be controlled by nutritional therapy and diuretics; and (v) patients with portal vein tumor thrombosis or extrahepatic metastasis. When four or more nodules were detected or the largest nodule was over 3 cm, RFA was preceded by transcatheter arterial chemoembolization (TACE) using epirubicin and gelatin sponge particles. Using combined examinations from ultrasonography and dynamic computed tomography (CT) scans or dynamic magnetic resonance imaging (MRI) or CT during angiography, diagnosis of HCC was confirmed in cases where the contrast pattern of the nodule in CT or MRI was hypervascular in the arterial phase and hypovascular in the portal phase. If the nodules were not consistent with typical contrast patterns for HCC, a needle biopsy of the tumor was taken for pathological diagnosis. The American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) 6th edition staging system for HCC was used for Tumor–Node–Metastasis (TNM) classification.18 The following three RF systems were used. From January 2000 to March 2000, 25 patients underwent RFA treatment using an RF 2000 generator system (Radio Therapeutics, Mountain View, CA, USA).

The remaining 461 patients (264 men, 197 women; age range 38–93 years; mean age 68.2 ± 8.7 years) were enrolled in this study. They consisted of 107 (23.2%) elderly subjects, aged PD0325901 mouse 75 years or over, and 354 non-elderly subjects. According to the estimate released by the National Cancer Center, Japan, the number of liver cancer mortalities in Japanese persons aged over 75 years increased, whereas that of subjects under 75 years decreased between 2004 and 2008.16 Additionally, the incidence of liver cancer continually increased in Japanese persons over 75 years

until 2005, whereas the incidence in persons under 75 years reached its peak in 2003.17 Therefore, we divided subjects into two groups (those <75 years and those ≥75 years) to analyze and discuss the strategy of treatment for elderly HCC patients. The indication for RFA treatment was that HCC consisted of five or fewer nodules, with each nodule having a maximum diameter of 30 mm, or that HCC consisted of a single tumor, regardless of size, and that hepatic function was not Child–Pugh grade C. CX-4945 cell line Radiofrequency ablation treatment was applied to cases (n = 226) that were not considered

to be suitable for resection for the following reasons: (i) impairment of liver function, and (ii) an excessive number of tumors or cardiopulmonary dysfunction. In addition, we applied RFA in cases (n = 235) where patients chose ablation therapy

even though surgery was also feasible. Exclusion criteria for RFA were: (i) MCE公司 total bilirubin concentration over 3 mg/dL; (ii) platelet count under 30 000/mm3; (iii) prothrombin activity under 50%; (iv) ascites that could not be controlled by nutritional therapy and diuretics; and (v) patients with portal vein tumor thrombosis or extrahepatic metastasis. When four or more nodules were detected or the largest nodule was over 3 cm, RFA was preceded by transcatheter arterial chemoembolization (TACE) using epirubicin and gelatin sponge particles. Using combined examinations from ultrasonography and dynamic computed tomography (CT) scans or dynamic magnetic resonance imaging (MRI) or CT during angiography, diagnosis of HCC was confirmed in cases where the contrast pattern of the nodule in CT or MRI was hypervascular in the arterial phase and hypovascular in the portal phase. If the nodules were not consistent with typical contrast patterns for HCC, a needle biopsy of the tumor was taken for pathological diagnosis. The American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) 6th edition staging system for HCC was used for Tumor–Node–Metastasis (TNM) classification.18 The following three RF systems were used. From January 2000 to March 2000, 25 patients underwent RFA treatment using an RF 2000 generator system (Radio Therapeutics, Mountain View, CA, USA).

The reason for the tissue-specific ARC-JNK interaction remains unclear. Because CARDs mediate protein-protein interactions and ARC’s CARD was shown to interact with Fas, FADD, procaspase-8, and Bax, its functional importance in Epigenetics inhibitor binding JNK was assessed.10 We disrupted ARC’s CARD by mutating two residues (L31F; G69R) that are conserved in death-fold proteins back to Ced-3.25 Mutant TAT-ARC abrogated the interaction of ectopic TAT-ARC with JNK1 and JNK2 and showed no protection against TNF-α-mediated liver failure (Fig. 7E,F). Thus, the CARD of ARC mediates its interaction with

JNK1 and JNK2. Thus, our results suggest that ARC inhibits JNK activation and translocation by a direct interaction between ARC’s CARD and JNK1 and JNK2. ARC is exceptional in its ability to antagonize both the extrinsic (death receptor) and the intrinsic (mitochondria / endoplasmic reticulum [ER]) death pathways.8-10 Here, we demonstrate highly efficient therapeutic in vivo delivery of ARC to the adult murine liver using the TAT protein transduction technique. Ectopic ARC delivery completely blocks Fas- and TNF-mediated hepatocyte apoptosis in vitro and in three different in vivo models of ALF protecting mice from death in preventive MK0683 and therapeutic settings. Fas-induced apoptosis is triggered by way of Fas receptor-mediated DISC assembly.4 TAT-ARC blocks caspase-8-dependent

cell killing by binding to members of the DISC, namely Fas, FADD, and procaspase-8. Additionally, it inhibits Fas-mediated Bax conformational activation and subsequent mitochondria-dependent death signaling. Hepatocytes are highly sensitive to Fas-induced apoptosis compared with other tissues and organs and absence of endogenous ARC might contribute to 上海皓元 this observation.2 Previous in vivo studies demonstrated successful hepatic delivery of small interfering RNA (siRNA) targeting Fas or caspase-8 of mice with Fas-mediated hepatitis.25, 26 However, the relevance of those therapeutic approaches targeting hepatocyte injury in ALF is limited due to its delayed mode of action and the low delivery efficiency of siRNA into hepatocytes.26,

27 TAT-ARC does not have these limitations and therefore might be a more valuable candidate for treatment of ALF in humans. Several studies have convincingly demonstrated a critical role of JNK during ConA or GalN/LPS-induced hepatocyte apoptosis.21, 28-30 These findings suggested JNK as a major therapeutic target and JNK-specific drugs are currently in clinical development. We demonstrate that administration of TAT-ARC prevents JNK activation in the liver upon ConA or GalN/LPS-induced hepatitis. In vitro experiments with recombinant JNK1 and JNK2 show binding with the CARD domain of ARC, indicating that ARC directly suppresses JNK activity, which has not been reported before. Traditionally, death-fold motifs use homotypic protein-protein interactions. The CARD of ARC engages in homotypic death-fold interactions as shown by ARC homodimerization.

We further investigated whether the MESIAH score can further classify survival of patients within each stratum of the BCLC staging system (Fig. 4). Whereas patients in BCLC stage 0 or A did well overall, there was a large degree of variability in survival of patients with BCLC stage B to D. In BCLC stage B, patients in the lowest quartile of the MESIAH score had 77% 3-year survival compared with those in highest quartile with 17% survival. The gap was even wider in BCLC stage C patients, in whom the 3-year survival in the lowest

and highest quartiles was 49% and 0%, respectively. Sensitivity analyses were performed to test the robustness of the MESIAH score. First we examined the effect of censoring patients at the time of liver transplantation (n = 133), as the Epigenetics inhibitor procedure may fundamentally affect the natural history of HCC. The result, however, was that censoring liver transplantation did not affect the overall performance of the model. MLN0128 The c-statistic in the derivation cohort was 0.79 and validation 0.81, which were again better than other staging systems (Supporting Table). Second, we repeated

the validation analysis excluding nonviral hepatitis patients in the validation cohort. Again, the result did not change materially (data not shown). In general, patient characteristics that determine the prognosis in HCC tend to be more complex than those for other solid tumors, as the vast majority of patients with HCC have underlying liver cirrhosis and the degree of hepatic dysfunction, in addition to the extent of the tumor, affects prognosis. This analysis demonstrates that the extent of the tumor as represented by variables such as the number and size of the lesions, vascular invasion, and extrahepatic metastasis and the underlying liver function, measured by MELD, are important independent predictors of survival in HCC patients.

Although this is not 上海皓元医药股份有限公司 the first observation that MELD may be useful in prognosticating patients with HCC,19 one of the advantages of the MESIAH score is that it only includes objective, reproducible variables. The value of an objectively quantifiable measure of disease has been appreciated with MELD, which has been rapidly become a common language among physicians globally. Similarly, we believe that the MESIAH score could be applied in retrospective studies or in epidemiologic research where nuanced details of clinical information are unavailable. We are reassured of the validity of the MESIAH score model, as it performed well in our cross-validation as well as in the independent dataset of our validation cohort. The c-statistic in the validation cohort was higher than that in the derivation cohort, indicating that it was easier for the score to separate HCC patients according to their prognosis in the former. This is likely attributed to the wider range of the score (and thus survival) with fewer patients receiving treatment that potentially alters the natural history in that cohort.

5, 13, 14 Zebrafish have also increasingly been used to model cancer.15 Liver tumors, generated after exposure to carcinogens, exhibit a stage-specific expression profile comparable to human HCC.6 These examples illustrate the increasing relevance and impact of the zebrafish to model liver disease. The study by Yin et al. also highlights the feasibility of performing chemical screens.

Due to the small size and high number of zebrafish embryos and larvae, thousands of animals can be exposed to chemicals in a single experiment. Here, the authors tested 338 compounds for their capacities to modulate HSC numbers. The power of this system notwithstanding, the necessity to use a different transgenic cell line remains: for the screen, the authors used another transgenic line, labeling wt1b-positive cells. The PI3K inhibitor expression of wt1b overlaps with hand2 in the liver but has not

been explicitly characterized in the report. This minor shortcoming underscores the remaining challenges of using in vivo fluorescent reporters for screening Selleck 5-Fluoracil purposes. This screen identified two retinoid receptor agonists with opposite effects on HSC formation, confirming the reported importance for retinoic acid in HSC formation. Taken together, these results demonstrate the potential to identify novel compounds that affect HSC number and activity in the zebrafish with direct therapeutic implications. What this model still needs to prove, however, is the identification

of novel signaling pathways affecting HSC MCE公司 formation and biology that have not been elucidated in other systems. Zebrafish have recently made the jump from the fish tank to the bedside, demonstrating our ability to discover novel therapeutics: a chemical genetic screening approach identified prostaglandin E2 as a novel regulator of hematopoietic stem cells.16 This research inspired translational work17 that led to a recently completed clinical phase 1 trial, which demonstrated the use of prostaglandin E2 treatment of umbilical cord blood stem cells prior to transplantation into patients with leukemia and lymphoma (NCT00890500).18 Similarly, current work in a zebrafish melanoma model will result in a soon-to-be-opened clinical trial19 (L. Zon, personal communication). Our work using (NCT01611675) the acetaminophen model combined with chemical genetic screening has also fostered the discovery of novel therapeutics and considerations for a clinical trial.12 Each of these examples underscores the growing relevance of the zebrafish in translational medicine. The study by Yin et al. represents a major step toward the use of the zebrafish model for many aspects of hepatology research: it opens the door for further studies into HSC activation and physiology in a tractable in vivo model.

Dilatation of the cerebral veins and venous sinuses may also selleck chemicals be a participatory mechanism and, in some situations, perhaps even the dominant mechanism. Some patients with stubborn orthostatic headaches, in recumbency, may report an earlier and a more effective relief in certain positions or postures, such as Trendelenburg position,[30] or by lying prone with the head dropped somewhat at the edge of the bed. It has been demonstrated that CSF OP is significantly higher in prone than in lateral decubitus position.[31] Headache, the most common clinical manifestation of spontaneous

CSF leaks, is often (although not always) associated with one or more of a variety of other manifestations listed in Table 2. Sometimes one or more of these may be the dominant clinical feature or, more rarely, the only clinical manifestation. Occasionally, headache may be completely absent. In the past two decades, increasing reports of various, and sometimes unexpected, manifestations

of spontaneous CSF leaks have appeared in the literature. Traction or compression Vincristine research buy is suspected to be the involved mechanism of various cranial nerve palsies in these patients. Cochleovestibular manifestations may result from traction or compression of the 8th cranial nerve or decrease in pressure of the perilymph, or both. Other manifestations have been similarly attributed to traction, compression, or displacement of various related structures including different lobes of the brain, brainstem or mesencephalon, pituitary stalk, or nerve roots.[32] Gait disorder and incontinence have been attributed by some researchers to spinal cord congestion. These attributions, however, are to be considered as proposed rather than proven mechanisms. In the early years

of MRI detection of pachymeningeal thickening, many patients were subjected to multiple CSF examinations in search of inflammatory, infectious, or neoplastic disease. Many lessons were learned including the substantial variability in the CSF findings in different patients with CSF leaks as well as in each individual MCE patient who had undergone multiple spinal taps on multiple occasions in the setting of symptomatic active CSF leaks. CSF OP is low in the large majority; but in a significant minority, perhaps in about one fourth of patients, it is within normal limits. The OP is uncommonly atmospheric and rarely is even negative. Color is often clear and only sometimes xanthochromic. Note that difficult and blood-tinged taps are not uncommon considering the very low pressure in some of the patients and presence of dilated epidural venous plexus in many (see spinal MRI findings and Table 4). Protein concentration may be normal or high. Values up to 100 mg/dL are not uncommon and concentrations as high as 1000 mg/dL have been reported.

15, 16 Accumulating evidence suggests that the formation of apoB100-BLp17 and apoB48-BLp18, 19 is accomplished sequentially. The two-step model

postulates that the initial product is a primordial particle, formed during apoB translation in the endoplasmic reticulum (ER). It is clear that microsomal TG transfer protein is involved in the early stage (first step) of apoB lipidation. However, the mechanism involved in the later stage (second step) is still not well understood. We have unexpectedly found that PLTP deficiency causes a significant impairment in hepatic secretion of VLDL.20 Likewise, it has been reported that animals overexpressing PLTP exhibit hepatic JQ1 ic50 VLDL overproduction.21 Associations of plasma PLTP activity with elevated apoB levels22 have been found in humans, as well. In a recent study, Masson et al.23 found that human PLTP transgenic rabbits showed a significant increase of BLp, but not of HDL cholesterol, in the circulation. These animals demonstrated increased atherosclerotic lesions after a high-fat diet feeding compared with controls. Alectinib manufacturer Nevertheless, the surprising discovery that PLTP

affects BLp secretion from the liver must be explored fully. The liver is one of the major sites of lipoprotein production and degradation, as well as PLTP expression. To investigate the role of PLTP in lipoproteins homeostasis, we initially planned to prepare a liver-specific KO mouse model using the Cre-Loxp system under the control of an albumin promoter. Finally, we created a unique mouse model that expresses PLTP acutely and specifically in the liver, with a PLTP-null background. We studied lipoprotein metabolism, VLDL secretion, and VLDL lipidation in these animals, concluding that liver PLTP-mediated VLDL production is one of the driving forces for plasma lipoprotein metabolism. AdV, adenovirus; apo, apolipoprotein; BLp, apoB-containing lipoproteins; CETP, 上海皓元医药股份有限公司 cholesteryl ester transfer protein; ER, endoplasmic reticulum;

FPLC, fast protein liquid chromatography; GFP, green fluorescent protein; HDL, high-density lipoprotein; KO, knockout; PCR, polymerase chain reaction; PERPP, post-ER presecretory proteolysis; PLTP, phospholipid transfer protein; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TG, triglycerides; TLC, thin-layer chromatography; VLDL, very low-density lipoprotein; WT, wild-type. To prepare PLTP-Flox mice, a 9.16-kb region used to construct the targeting vector was first subcloned from a positively identified C57BL/6 (RPCI23) BAC clone. The region was designed so that the short homology arm extended about 1.68 kb (3′ to exon 3), and the long homology arm extended about 6.85 kb (5′ to exon 2). The loxP and FRT double-flanked Neo cassette was inserted on the 3′ side of exon 3, and the single loxP site was inserted at the 5′ side of exon 2. (Fig. 1A). All mice used in this study were aged 12-16 weeks, with a C57BL/6J genetic background.