Genetic tools for the stable overexpression of circular RNAs.

Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

A Versatile ES Cell-Based Melanoma Mouse Modeling Platform.

The cumbersome and time-consuming process of generating new mouse strains and multiallelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. The platform incorporates 12 clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, and Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes of interest. The ESCs produce high-contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using the ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlighted the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, complementary genetic methods demonstrated the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we showed that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo Thus, when combined with sophisticated genetic tools, the ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.Significance: This study presents a high-throughput and versatile ES cell-based mouse modeling platform that can be combined with state-of-the-art genetic tools to address unanswered questions in melanoma in vivo See related commentary by Thorkelsson et al., p. 655.

Utilising the resources of the International Knockout Mouse Consortium: the Australian experience.

Mouse models play a key role in the understanding gene function, human development and disease. In 2007, the Australian Government provided funding to establish the Monash University embryonic stem cell-to-mouse (ES2M) facility. This was part of the broader Australian Phenomics Network, a national infrastructure initiative aimed at maximising access to global resources for understanding gene function in the mouse. The remit of the ES2M facility is to provide subsidised access for Australian biomedical researchers to the ES cell resources available from the International Knockout Mouse Consortium (IKMC). The stated aim of the IKMC is to generate a genetically modified mouse ES cell line for all of the ~23,000 genes in the mouse genome. The principal function of the Monash University ES2M service is to import genetically modified ES cells into Australia and to convert them into live mice with the potential to study human disease. Through advantages of economy of scale and established relationships with ES cell repositories worldwide, we have created over 110 germline mouse strains sourced from all of the major ES providers worldwide. We comment on our experience in generating these mouse lines; providing a snapshot of a "clients" perspective of using the IKMC resource and one which we hope will serve as a guide to other institutions or organisations contemplating establishing a similar centralised service.

Epithelial NEMO links innate immunity to chronic intestinal inflammation.

Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease. The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis-acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides. However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF-kappaB, a master regulator of pro-inflammatory responses, functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-kappaB through conditional ablation of NEMO (also called IkappaB kinase-gamma (IKKgamma)) or both IKK1 (IKKalpha) and IKK2 (IKKbeta)-IKK subunits essential for NF-kappaB activation-spontaneously caused severe chronic intestinal inflammation in mice. NF-kappaB deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor (TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-kappaB signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract, causing an inflammatory-bowel-disease-like phenotype. Our results identify NF-kappaB signalling in the gut epithelium as a critical regulator of epithelial integrity and intestinal immune homeostasis, and have important implications for understanding the mechanisms controlling the pathogenesis of human inflammatory bowel disease.

Normal epidermal differentiation but impaired skin-barrier formation upon keratinocyte-restricted IKK1 ablation.

The kinase IKK1 (also known as IKKalpha) was previously reported to regulate epidermal development and skeletal morphogenesis by acting in keratinocytes to induce their differentiation in an NF-kappaB independent manner. Here, we show that mice with epidermal keratinocyte-specific IKK1 ablation (hereafter referred to as IKK1(EKO)) develop a normally differentiated stratified epidermis, demonstrating that the function of IKK1 in inducing epidermal differentiation is not keratinocyte-autonomous. Despite normal epidermal stratification, the IKK1(EKO) mice display impaired epidermal-barrier function and increased transepidermal water loss, due to defects in stratum corneum lipid composition and in epidermal tight junctions. These defects are caused by the deregulation of retinoic acid target genes, encoding key lipid modifying enzymes and tight junction proteins, in the IKK1-deficient epidermis. Furthermore, we show that IKK1-deficient cells display impaired retinoic acid-induced gene transcription, and that IKK1 is recruited to the promoters of retinoic acid-regulated genes, suggesting that one mechanism by which IKK1 controls epidermal-barrier formation is by regulating the expression of retinoic acid receptor target genes in keratinocytes.

Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma.

The IkappaB kinase (IKK) subunit NEMO/IKKgamma is essential for activation of the transcription factor NF-kappaB, which regulates cellular responses to inflammation. The function of NEMO in the adult liver remains elusive. Here we show that ablation of NEMO in liver parenchymal cells caused the spontaneous development of hepatocellular carcinoma in mice. Tumor development was preceded by chronic liver disease resembling human nonalcoholic steatohepatitis (NASH). Antioxidant treatment and genetic ablation of FADD demonstrated that death receptor-mediated and oxidative stress-dependent death of NEMO-deficient hepatocytes triggered disease pathogenesis in this model. These results reveal that NEMO-mediated NF-kappaB activation in hepatocytes has an essential physiological function to prevent the spontaneous development of steatohepatitis and hepatocellular carcinoma, identifying NEMO as a tumor suppressor in the liver.

Skin lesion development in a mouse model of incontinentia pigmenti is triggered by NEMO deficiency in epidermal keratinocytes and requires TNF signaling.

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase, is essential for NF-kappaB activation. Mutations disrupting the X-linked NEMO gene cause incontinentia pigmenti (IP), a human genetic disease characterized by male embryonic lethality and by a complex pathology affecting primarily the skin in heterozygous females. The cellular and molecular mechanisms leading to skin lesion pathogenesis in IP patients remain elusive. Here we used epidermis-specific deletion of NEMO in mice to investigate the mechanisms causing the skin pathology in IP. NEMO deletion completely inhibited NF-kappaB activation and sensitized keratinocytes to tumor necrosis factor (TNF)-induced death but did not affect epidermal development. Keratinocyte-restricted NEMO deletion, either constitutive or induced in adult skin, caused inflammatory skin lesions, identifying the NEMO-deficient keratinocyte as the initiating cell type that triggers the skin pathology in IP. Furthermore, genetic ablation of tumor necrosis factor receptor 1 (TNFRI) rescued the skin phenotype demonstrating that TNF signaling is essential for skin lesion pathogenesis in IP. These results identify the NEMO-deficient keratinocyte as a potent initiator of skin inflammation and provide novel insights into the mechanism leading to the pathogenesis of IP.

Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein.

Three mouse lines expressing Cre recombinase under the control of the human K14 promoter induced specific deletion of loxP flanked target sequences in the epidermis, in tongue, and thymic epithelium of the offspring where the Cre allele was inherited from the father. Where the mother carried the Cre allele, loxP flanked sequences were completely deleted in all tissues of the offspring, even in littermates that did not inherit the Cre allele. This maternally inherited phenotype indicates that the human K14 promoter is transcriptionally active in murine oocytes and that the enzyme remains active until after fertilization, even when the Cre allele becomes transmitted to the polar bodies during meiosis. Detection of K14 mRNA by RT-PCR in murine ovaries and immunohistochemical identification of the K14 protein in oocytes demonstrates that the human K14 promoter behaves like its murine homolog, thus identifying K14 as an authentic oocytic protein.

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2.

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.

Negative regulation of cell growth and differentiation by TSG101 through association with p21(Cip1/WAF1).

TSG101 was discovered in a screen for tumor susceptibility genes and has since been shown to have a multiplicity of biological effects. However, the basis for TSG101's ability to regulate cell growth has not been elucidated. We report here that the TSG101 protein binds to the cyclin/cyclin-dependent kinase (CDK) inhibitor (CKI) p21(Cip1/WAF1) and increases stability of the p21 protein in HEK293F cells and differentiating primary keratinocytes, suppressing differentiation in a p21-dependent manner. In proliferating keratinocytes where the p21 protein is relatively stable, TSG101 does not affect the stability or expression of p21 but shows p21-dependent recruitment to cyclin/CDK complexes, inhibits cyclin/CDK activity, and causes strong growth suppression to a much greater extent in p21+/+ than in p21-/- cells. Conversely, suppression of endogenous TSG101 expression by an antisense TSG101 cDNA causes doubling of the fraction of keratinocytes in the S phase of the cell cycle as occurs during p21 deficiency. Our results indicate that TSG101 has a direct role in the control of growth and differentiation in primary epithelial cells, and that p21 is an important mediator of these TSG101 functions.