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The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras[sup.MUT] levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous Kras[sup.MUT] in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgf-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer., Author(s): Sebastian Mueller [1, 2]; Thomas Engleitner [1, 2, 3]; Roman Maresch [1, 2, 3]; Magdalena Zukowska [1, 2]; Sebastian Lange [1, 2]; Thorsten Kaltenbacher [1, 2, 3]; Bjrn Konukiewitz [...]

Author(s): Dieter Egli [sup.1] , Michael V. Zuccaro [sup.2] , Michael Kosicki [sup.3] , George M. Church [sup.4] , Allan Bradley [sup.3] , Maria Jasin [sup.5] Author Affiliations:(1) Department of [...]

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation., Pancreatic ductal adenocarcinoma (PDAC) is a fearsome disease, with a mortality rate >95% that has remained unchanged for decades (1). GEMMs that faithfully recapitulate the histological, molecular, genetic and clinical [...]

Author(s): Mary E. Dickinson; Ann M. Flenniken; Xiao Ji; Lydia Teboul; Michael D. Wong; Jacqueline K. White; Terrence F. Meehan; Wolfgang J. Weninger; Henrik Westerberg; Hibret Adissu; Candice N. Baker; [...]

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders (1-4). However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications (3,5). The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome (6). Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) (7) and piggyBac (8,9) technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the [α.sub.1]-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for [α.sub.1]-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies., At present, available methods for gene targeting rely on positive selection to isolate rare clones that have undergone homologous recombination. To remove the unwanted selection cassettes, Cre/loxP or Flp/FRT recombination [...]

Letter to the Editor, Author(s): Matt Truppo, George Yancopoulos, Andrew J. Murphy, Allan Bradley, Larry Green, Ivo Lorenz Author Affiliations: Non-animal-derived antibodies: pharma companies respond We disagree with claims by Alison Gray and colleagues [...]

Author(s): Michael C. Zody (corresponding author) [1]; Manuel Garber [1]; David J. Adams [2]; Ted Sharpe [1]; Jennifer Harrow [2]; James R. Lupski [3]; Christine Nicholson [2]; Steven M. Searle [...]

Author(s): Ge Guo [1]; Wei Wang [1]; Allan Bradley (corresponding author) Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice [...]

Author(s): Benjamin T. Kile [1, 4]; Kathryn E. Hentges [1, 4]; Amander T. Clark [1, 4]; Hisashi Nakamura [1]; Andrew P. Salinger [1]; Bin Liu [1]; Neil Box [1]; David [...]

Author(s): Stuart D. Tyner [1, 2]; Sundaresan Venkatachalam [2, 3]; Jene Choi [3]; Stephen Jones [4]; Nader Ghebranious [5]; Herbert Igelmann [6]; Xiongbin Lu [3]; Gabrielle Soron [3]; Benjamin Cooper [...]

Author(s): Elizabeth A. Lindsay [1]; Francesca Vitelli [1]; Hong Su [2]; Masae Morishima [1]; Tuong Huynh [1]; Tiziano Pramparo [1]; Vesna Jurecic [3]; George Ogunrinu [4]; Helen F. Sutherland [5]; [...]

It has been possible to identify the pathogenic basis of the most clinically severe element of DiGeorge syndrome, the most common deletion syndrome in humans. Heterozygously deleted mice with cardiovascular abnormalities of the same type as those connected with the band 22q11 are a model of the most significant clinical finding in DiGeorge syndrome. They also offer information about the embryological mechanisms on which congenital heart disease in DiGeorge syndrome patients is based.

mPer2, a mammalian homologue of the Drosophila circadian clock gene period, has been shown to encode a dedicated element of the mammalian circadian clock. Mice homozygous for this mutation demonstrate a shorter circadian period followed by a loss of circadian rhythmicity in constant darkness. mPer2 is probably a central clock element, as mutation in mPer2 brings about a change in the expression of other genes thought to be connected with the clock.

The function of p63, a homologue of the p53 tumour suppressor, during embryogenesis has been investigated by mutating the p63 gene using embryonic stem cell technology. It was established that p63-deficient newborn mice demonstrate clear limb and skin defects. The downstream targets of p63 probably involve direct or indirect activation of factors associated with ectodermal-mesenchymal communication, which are needed for morphogenesis of the tooth, hair follicle, apical ectodermal ridge and mammary bud.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts., Author(s): Mary E. Dickinson [1]; Ann M. Flenniken [2, 3]; Xiao Ji [4]; Lydia Teboul [5]; Michael D. Wong [2, 6]; Jacqueline K. White [7]; Terrence F. Meehan [8]; Wolfgang [...]

People with mutations in the BRCA1 or BRCA2 genes have a dominant predisposition to cancer, with inherited mutations in BRCA1 causing breast and ovarian cancer and in BRCA2 mostly breast cancer. Recent observations of a link between BRCA1 and HsRad51, give some insight into the BRCA1 function. The function of BRCA2 in mouse cell proliferation and possible DNA repair is described, concentrating on the developmental arrest in BRCA2-deficient embryos, and their radiation sensitivity.

Immunohistochemistry and in situ hybridization were performed to investigate the role of Hoxb-1 gene in the organization of motor neurons in mice. Hoxb-1 has been known to be expressed in rhombomere 4 (r4) and the results of the experiment confirmed that its absence causes changes in the identity of r4. The results further showed that Hoxb-1 plays the role of maintaining rhombomere identity by controlling the migratory properties of motor neurons in the hindbrain.

Paraxial mesoderm cells fail to form epithelia in mice homozygous for a paraxis null mutation that results in disruption of somite formation. Paraxis is a helix-loop-helix transcription factor expressed in paraxial cells and somites. The absence of normal somites gives rise to improper patterns in axial skeleton and skeletal muscles. The segmentation of the embryo is independent of the formation of epithelial somites. Thus, paraxis regulates somite morphogenesis in mice embryos.

Osteocalcin-deficient mice develop a phenotype with bones of higher mass and better functional quality. Histomorphometric analysis of these mice, before and after ovariectomy, reveals increased bone formation without any alteration in bone resorption or mineralization. The functional molecular mechanism for osteocalcin-controlled bone matrix deposition is unknown. Osteocalcin is a non-collagenous protein that binds to a specific receptor and functions as a negative controler of bone formation.

Large deletions, duplications and inversions can be designed in embryonic stem cells in mice. The method involves the Cre protein, an enzyme that catalyzes the recombination between loxP sites. Consecutive targeting of loxP recombination substrates followed by recombination stimulated by Cre produces defined deficiencies. Deletions to the tune of 90 kb to 3-4 cM, which serve as a tool for screening of recessive mutations, are possible. The availability of mice with segmental haploidy will be useful in genetic screens which will help develop models of human chromosomal diseases.

A study of mice with mutated cells found that those that were homozygous for the xerodema pigmentosa Group C mutant allele had a higher incidence of tumor growth than a control population or mice heterozygous for the mutant allele. The mice were mutated using embryonic stem cell technology.

A null mutation in the Act RcII gene in mice is generated utilizing embryonic stem cell technology to study the function of the ActRcII in mammalian reproduction and development. The mice with ActRcII deficiency exhibited facial and skeletal abnormalities identical to the Pierre-Robin syndrome in humans. But the abnormalities did not affect the growth and development of these mice. This indicates that during ActRcII absence other hormones control the growth and development.

The function of follistatin in mammals is investigated by using the loss-of-function mutant mice. The absence of follistatin in mice reveals a retardation in growth and a reduction in the diaphragm and intercostal muscle mass. The shiny taut skin, skeletal defects in the ribs and abnormal tooth development are other observed features. These indicate that follistatin has more influence on the growth and regulation of a mammal than activin.

A muscle specific transcription factor that can induce myogenesis in a variety of cell types is myogenin. To test myogenin's role in embryonic muscle development, homozygous mice were generated for a targeted mutation in the myogenin gene. Though these mice survived fetal development, they were born immobile and died immediately after birth. They have severe reduction in their skeletal muscle throughout their body. Myogenin is thus an essential component for the skeletal muscle formation during embryogenesis. Mice lacking myogenin have gross rib defects, which are not revealed at the cellular level as bone and cartilage appear histologically normal.

Alpha-inhibin, the gene for one of a group of dimeric growth factors, has been identified as a tumor-suppressor gene. The development of gonadal tumors in mice that were bred to lack the alpha-inhibin gene confirmed the gene's role in preventing the uncontrolled growth of cells during the embryonic phase. The alpha-inhibin-deficient mice were produced using homologous recombination in embryonic stem cells.

Mouse embryos lacking the nuclear phosphoprotein (Rb) for the retinoblastoma (RB) tumor-suppressor gene die before the 16th embryonic day and display an array of genetic defects while alive. These defects affect the hematopoietic system which produces an abnormal proliferation of immature nucleated erythrocytes and the nervous system where ectopic mitoses and massive cell death occur. The finding that injecting human RB mini-transgene into the mutant mice rectifies the abnormalities proves that Rb is required for normal embryonic growth in mice.

Author(s): Allan Bradley [1] [illus. 1] The mouse genome sequence, published in this issue [1], has already made a huge impact on the research community. Although only a draft, it [...]

The Mdm2 proto-oncogene regulates the p53 activity during the embryonic development. This is probably because of the activity of p53 in the absence of Mdm2, which leads to the arrest of the cell cycle at the G1 stage during embryo development. On the other hand, the Mdm2/p53 null mutants show normal morphogenesis. A complex between the Mdm2 oncoprotein and the p53 tumor-suppressor protein is responsible for p53-mediated transregulation of the gene expression.

Mice with mutations in activin-beta A or in beta A and beta A are generated to investigate the function of mammalian activins in vivo. The activins are known to be dimeric members of the transforming growth superfamily. They are widely expressed during the development of murine. The activin is essential for the survival and complete development of an individual animal.

Author(s): Louise van der Weyden; Jos Jonkers; Allan Bradley (corresponding author) Several inherited human diseases are characterized by the formation of polyps in the gut. Polyps are benign outgrowths of [...]

Author(s): Milind Suraokar (corresponding author) [1]; Allan Bradley [2] Over the past few thousand years, the physical characteristics of sheep and their underlying genetic make-up have been slowly altered by [...]

Modelling human diseases caused by large genomic rearrangements has become more accessible since the utilization of CRISPR/Cas9 in mammalian systems. In a previous study, we showed that genomic rearrangements of up to one million base pairs can be generated by direct injection of CRISPR/Cas9 reagents into mouse zygotes. Although these rearrangements are ascertained by junction PCR, we describe here a variety of anticipated structural changes often involving reintegration of the region demarcated by the gRNAs in the vicinity of the edited locus. We illustrate here some of this diversity detected by high-resolution fibre-FISH and conclude that extensive molecular analysis is required to fully understand the structure of engineered chromosomes generated by Cas9.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.

Aneuploidy leads to severe developmental defects in mammals and is also a hallmark of cancer. However, whether aneuploidy is a driving cause or a consequence of tumor formation remains controversial. Paradoxically, existing studies based on aneuploid yeast and mouse fibroblasts have shown that aneuploidy is usually detrimental to cellular fitness. Here, we examined the effects of aneuploidy on mouse embryonic stem (ES) cells by generating a series of cell lines that each carries an extra copy of single chromosomes, including trisomy 6, 8, 11, 12, or 15. Most of these aneuploid cell lines had rapid proliferation rates and enhanced colony formation efficiencies. They were less dependent on growth factors for self-renewal and showed a reduced capacity to differentiate in vitro Moreover, trisomic stem cells formed teratomas more efficiently, from which undifferentiated cells can be recovered. Further investigations demonstrated that co-culture of wild-type and aneuploid ES cells or supplementation with extracellular BMP4 rescues the differentiation defects of aneuploid ES cells., (© 2016 The Authors.)

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

Most tumour suppressor genes (TSGs) have been found through linkage studies in cancer predisposed families where the mutations have a high penetrance, for example, the breast cancer genes BRCA1 and BRCA2. Loss of heterozygosity (LOH) analyses of sporadic breast tumours indicate that there are many other putative TSGs yet to be identified. One such locus is proximal to BRCA1 on human chromosome 17q21. In an attempt to isolate this putative TSG, we have assessed a portion of the orthologous region on mouse chromosome 11 for its tumorigenic potential using segmental haploidy in combination with a p53 mutation. Two populations of animals were studied, with the deleted region being either on the same (cis) or on the homologous chromosome (trans) to a targeted mutant p53 allele. The deficiency elevated the tumour susceptibility of p53 heterozygous mice and modified the tumour spectrum, but only when the deficiency was in trans with the p53 mutation. Even though the genotype of these mice is identical, allelic phasing affects both the tumour spectrum and progression.