Frank Schnütgen

A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome

A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration “hot spots.” Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.

EphA4-Dependent Axon Guidance Is Mediated by the RacGAP α2-Chimaerin

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. ...

Splinkerette PCR for more efficient characterization of gene trap events

NOTE: In the version of this article initially published, the second author (Jens Hansen) should have been listed as an equal contributor with the first author. The last two authors (Harald von Melchner and Patricia Ruiz Noppinger) should have been listed as corresponding authors. The error has been corrected in the HTML and PDF versions of the article.

Engineering Embryonic Stem Cells with Recombinase Systems

The combined use of site-specific recombination and gene targeting or trapping in embryonic stem cells (ESCs) has resulted in the emergence of technologies that enable the induction of mouse mutations in a prespecified temporal and spatially restricted manner. Their large-scale implementation by several international mouse mutagenesis programs will lead to the assembly of a library of ES cell lines harboring conditional mutations in every single gene of the mouse genome. In anticipation of this unprecedented resource, this chapter will focus on site-specific recombination strategies and issues pertinent to ESCs and mice. The upcoming ESC resource and the increasing sophistication of site-specific recombination technologies will greatly assist the functional annotation of the human genome and the animal modeling of human disease.

Adopting the good reFLEXes when generating conditional alterations in the mouse genome

Major advances have been made in the use of the Cre/loxP system for conditional gene targeting in the mouse. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending upon the orientation of the flanking loxP sites, and the use of wild-type loxP and variant lox511 sites, we devised an efficient and reliable Cre-mediated genetic switch, called FLEX, through which expression of a given gene can be turned off, while expression of another one can be simultaneously turned on. We discuss how this innovative, flexible and powerful approach, which virtually adapts to any kind of site-specific recombinase (e.g., Cre and Flp recombinases), can be used to easily generate, even at high throughput and genome wide scale, many genetic modifications in a conditional manner, including those which were considered as difficult or impossible to achieve.

High-throughput trapping of secretory pathway genes in mouse embryonic stem cells

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, ∼66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (), are freely available to the scientific community.

Single-Stranded DNA-Binding Transcriptional Regulator FUBP1 Is Essential for Fetal and Adult Hematopoietic Stem Cell Self-Renewal.

The ability of hematopoietic stem cells (HSCs) to self-renew is a prerequisite for the establishment of definitive hematopoiesis and life-long blood regeneration. Here, we report the single-stranded DNA-binding transcriptional regulator far upstream element (FUSE)-binding protein 1 (FUBP1) as an essential factor of HSC self-renewal. Functional inactivation of FUBP1 in two different mouse models resulted in embryonic lethal anemia at around E15.5 caused by severely diminished HSCs. Fetal and adult HSCs lacking FUBP1 revealed an HSC-intrinsic defect in their maintenance, expansion, and long-term blood reconstitution, but could differentiate into all hematopoietic lineages. FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule. These changes caused an increase in generation time and death of HSCs as determined by video-microscopy-based tracking. Our data establish FUBP1 and its recognition of single-stranded genomic DNA as an important element in the transcriptional regulation of HSC self-renewal.

Conditional Gene Trapping Using the FLEx System

The knowledge about the complete genome sequences of mouse, human, and other organisms is only the first step toward the functional annotation of all genes. It facilitates the recognition of sequence conservation, which helps to distinguish between important and not important and also coding from noncoding sequence. Nevertheless, approximately only 50% of all mouse genes have been entirely annotated to date. In the postgenomic era, large-scale projects have been initiated to describe also the expression (Emap, Eurexpress) and the function (International Gene Trap Consortium, Eucomm, Norcomm, Komp) of all mouse genes. By building up on these resources, the average amount of time starting from a gene-coding sequence to finally studying its function in a living organism or embryo, has shortened significantly within the last decade. Several recent developments, namely, in bioinformatics and gene synthesis but also in targeted and random mutagenesis have contributed to the current status. This chapter will highlight the milestones that have been undertaken in order to saturate the mouse genome with gene trap mutations. We have no intention to cover the entire field but will instead focus on most recent vectors and protocols, which have turned out to be most useful in order to promote the technology. Therefore, we apologize upfront to the many studies that could not be mentioned here solely owing to space limitations but which nevertheless made significant contributions to our current understanding. This chapter will finally provide guidance on possible uses of conditional gene trap alleles as well as detailed protocols for the application of this recent technology.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays

Abstract Measuring NADPH oxidase (Nox)‐derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH‐stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1‐Nox2‐Nox4‐/‐) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH‐stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH‐stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH‐stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH‐dependent signal in assays utilizing lucigenin, L‐012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR‐/‐) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR‐/‐ abolished the signal in NADPH‐stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH‐dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays. Graphical abstract Figure. No Caption available. HighlightsNox activity of intact cells could not be recapitulated in membranes treated with NADPH.Proteomics of membranes show P450 reductase as source of NADPH‐dependent signal.Microsomes overexpressing Cytochrome P450 system produce a NADPH‐dependent signal.Knockout of P450 reductase (CRISPR/Cas9) abolished lucigenin signal in HEK cell membranes.Knockout of POR but not p22phox abolishes the basal and Angiotensin II‐stimulated NADPH‐dependent signal in SMC membranes.

Hoxa9 and Meis1 cooperatively induce addiction to syk signaling by suppressing miR-146a in acute myeloid leukemia

Summary The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.