P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology facilitates somatic genome editing to reveal cooperative genetic interactions at the cellular level without extensive breeding between different mutant animals. Here we propose a transgenic inducible Cas9 effector-CRISPR mutagen ( ICE CRIM) mouse model in which CRISPR/Cas9-mediated somatic mutagenesis events can occur in response to Cre expression. The well-known tumor suppressor gene, Trp53, and 2 important DNA mismatch repair genes, Mlh1 and Msh2, were selected to be our somatic mutagenesis targets. Amplicon-based sequencing was performed to validate the editing efficiency and to identify the mutant allelic series. Crossed with various Cre lines, the Trp53 ICE CRIM alleles were activated to generate targeted cancer gene somatic or germ line mutant variants. We provide experimental evidence to show that an activated ICE CRIM can mutate both targeted alleles within a cell. Simultaneous disruption of multiple genes was also achieved when there were multiple single-guide RNA expression cassettes embedded within an activated ICE CRIM. Our mouse model can be used to generate mutant pools in vivo, which enables a functional screen to be performed in situ. Our results also provide evidence to support a monoclonal origin of hematopoietic neoplasms and to indicate that DNA mismatch repair deficiency accelerates tumorigenesis in Trp53 mutant genetic background.-Fan, H.-H., Yu, I.-S., Lin, Y.-H., Wang, S.-Y., Liaw, Y.-H., Chen, P.-L., Yang, T.-L., Lin, S.-W., Chen, Y.-T. P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.

Functional and structural deficits of the dentate gyrus network coincide with emerging spontaneous seizures in an Scn1a mutant Dravet Syndrome model during development.

Dravet syndrome (DS) is characterized by severe infant-onset myoclonic epilepsy along with delayed psychomotor development and heightened premature mortality. A primary monogenic cause is mutation of the SCN1A gene, which encodes the voltage-gated sodium channel subunit Nav1.1. The nature and timing of changes caused by SCN1A mutation in the hippocampal dentate gyrus (DG) network, a core area for gating major excitatory input to hippocampus and a classic epileptogenic zone, are not well known. In particularly, it is still not clear whether the developmental deficit of this epileptogenic neural network temporally matches with the progress of seizure development. Here, we investigated the emerging functional and structural deficits of the DG network in a novel mouse model (Scn1a(E1099X/+)) that mimics the genetic deficit of human DS. Scn1a(E1099X/+) (Het) mice, similarly to human DS patients, exhibited early spontaneous seizures and were more susceptible to hyperthermia-induced seizures starting at postnatal week (PW) 3, with seizures peaking at PW4. During the same period, the Het DG exhibited a greater reduction of Nav1.1-expressing GABAergic neurons compared to other hippocampal areas. Het DG GABAergic neurons showed altered action potential kinetics, reduced excitability, and generated fewer spontaneous inhibitory inputs into DG granule cells. The effect of reduced inhibitory input to DG granule cells was exacerbated by heightened spontaneous excitatory transmission and elevated excitatory release probability in these cells. In addition to electrophysiological deficit, we observed emerging morphological abnormalities of DG granule cells. Het granule cells exhibited progressively reduced dendritic arborization and excessive spines, which coincided with imbalanced network activity and the developmental onset of spontaneous seizures. Taken together, our results establish the existence of significant structural and functional developmental deficits of the DG network and the temporal correlation between emergence of these deficits and the onset of seizures in Het animals. Most importantly, our results uncover the developmental deficits of neural connectivity in Het mice. Such structural abnormalities likely further exacerbate network instability and compromise higher-order cognitive processing later in development, and thus highlight the multifaceted impacts of Scn1a deficiency on neural development.

Wdhd1 is essential for early mouse embryogenesis.

WD repeat and HMG-box DNA binding protein 1 (Wdhd1) is the mouse ortholog of budding yeast Chromosome Transmission Fidelity 4 (CTF4), the protein product of which integrates the MCM2-7 helicase and DNA polymerase α/primase complex to initiate DNA replication. Previous work in fruit flies, Xenopus egg extracts, and human cell lines suggest that Wdhd1 is required for efficient DNA synthesis. However, rigorous in vivo functional studies on Wdhd1 in mammals are unavailable. In the present study, we have successfully generated a Wdhd1 null allele in mice through CRISPR/Cas9-mediated genome editing to investigate the role of Wdhd1 in embryogenesis in vivo. We characterized Wdhd1 expression using quantitative reverse-transcription polymerase chain reaction, and assessed embryonic cell proliferation by histology in both pre- and peri-implantation embryos. While Wdhd1 heterozygous mutant mice were grossly normal and fertile, we observed a reduction in cell proliferation by the gastrulation stage in Wdhd1 homozygous null mutant embryos which severely hampered their growth and viability. These results indicate that Wdhd1 plays a major role in cell proliferation during embryogenesis in mice.

Growth arrest DNA damage-inducible gene 45 gamma expression as a prognostic and predictive biomarker in hepatocellular carcinoma.

Growth arrest DNA damage-inducible gene 45 (GADD45) family proteins play a crucial role in regulating cellular stress responses and apoptosis. The present study explored the prognostic and predictive role of GADD45γ in hepatocellular carcinoma (HCC) treatment. GADD45γ expression in HCC cells was examined using quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. The control of GADD45γ transcription was examined using a luciferase reporter assay and chromatin immunoprecipitation. The in vivo induction of GADD45γ was performed using adenoviral transfer. The expression of GADD45γ in HCC tumor tissues from patients who had undergone curative resection was measured using qRT-PCR. Sorafenib induced expression of GADD45γ mRNA and protein, independent of its RAF kinase inhibitor activity. GADD45γ induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6-7 µM) than in sorafenib-resistant HCC cells (Hep3B, Huh-7R, and HepG2R, IC50 12-15 µM). Overexpression of GADD45γ reversed sorafenib resistance in vitro and in vivo, whereas GADD45γ expression knockdown by using siRNA partially abrogated the proapoptotic effects of sorafenib on sorafenib-sensitive cells. Overexpression of survivin in HCC cells abolished the antitumor enhancement between GADD45γ overexpression and sorafenib treatment, suggesting that survivin is a crucial mediator of antitumor effects of GADD45γ. GADD45γ expression decreased in tumors from patients with HCC who had undergone curative surgery, and low GADD45γ expression was an independent prognostic factor for poor survival, in addition to old age and vascular invasion. The preceding data indicate that GADD45γ suppression is a poor prognostic factor in patients with HCC and may help predict sorafenib efficacy in HCC.

A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

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.

Induction of DNA damage-inducible gene GADD45beta contributes to sorafenib-induced apoptosis in hepatocellular carcinoma cells.

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45ß (GADD45ß), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45ß expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45ß mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45ß induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45ß induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45ßor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45ß promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45ß induction by sorafenib. Together, our findings suggest that GADD45ß induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.

Induction of Bim expression contributes to the antitumor synergy between sorafenib and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor CI-1040 in hepatocellular carcinoma.

PURPOSE: Sorafenib has proved survival benefit for patients with advanced hepatocellular carcinoma (HCC). This study explored whether the efficacy of sorafenib can be improved by adding the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor CI-1040 to vertically block the Raf/MEK/ERK pathway. EXPERIMENTAL DESIGN: The growth inhibitory effects of sorafenib and CI-1040 were tested in HCC cell lines (Huh-7 and Hep3B) and human umbilical vascular endothelial cells (HUVEC). The potential synergistic growth inhibitory effects were measured by median effect analysis. Apoptosis was measured by flow cytometry. The effects on ERK phosphorylation and levels of apoptosis regulatory proteins were measured by Western blotting. The in vivo antitumor activity of sorafenib and CI-1040 were tested in xenograft HCC models. RESULTS: Combination of sorafenib and CI-1040 synergistically inhibited ERK phosphorylation and cell growth and induced apoptosis in both HCC cells and HUVECs. Increased expression of Bim protein, which correlated with the extent of ERK inhibition, was found in both HCC cells and HUVECs. Knockdown of Bim expression by small interfering RNA partially abrogated the synergistic proapoptotic effects of sorafenib and CI-1040. Combination therapy inhibited tumor growth significantly better than either single agent in the xenograft models. CONCLUSION: The antitumor effects of sorafenib in HCC can be improved by vertical blockade of Raf/MEK/ERK signaling with CI-1040.