Benedikt Wefers

Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms.

Generating Conditional Knockout Mice

Gene targeting in ES cells is extensively used to generate designed mouse mutants and to study gene function in vivo. Knockout mice that harbor a null allele in their germline provide appropriate genetic models of inherited diseases and often exhibit embryonic or early postnatal lethality. To study gene function in adult mice and in selected cell types, a refined strategy for conditional gene inactivation has been developed that relies on the DNA recombinase Cre and its recognition (loxP) sites. For conditional mutagenesis, a target gene is modified by the insertion of two loxP sites that enable to excise the flanked (floxed) gene segment through Cre-mediated recombination. Conditional mutant mice are obtained by crossing the floxed strain with a Cre transgenic line such that the target gene becomes inactivated in vivo within the expression domain of Cre. A large collection of Cre transgenic lines has been generated over time and can be used in a combinatorial manner to achieve gene inactivation in many different cell types. A growing number of CreER(T2) transgenic mice further allows for inducible inactivation of floxed alleles in adult mice upon administration of tamoxifen. This chapter covers the design and construction of loxP flanked alleles and refers to the vectors, ES cells, and mice generated by the European conditional mouse mutagenesis (EUCOMM) project. We further describe the design and use of Cre and CreER(T2) transgenic mice and a convenient breeding strategy to raise conditional mutants and controls for phenotype analysis.

Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA

Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator–like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2–3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.

The FTD‐like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism

Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk for several neurodegenerative diseases including Alzheimers disease and frontotemporal dementia (FTD). Homozygous TREM2 missense mutations, such as p.T66M, lead to the FTD‐like syndrome, but how they cause pathology is unknown. Using CRISPR/Cas9 genome editing, we generated a knock‐in mouse model for the disease‐associated Trem2 p.T66M mutation. Consistent with a loss‐of‐function mutation, we observe an intracellular accumulation of immature mutant Trem2 and reduced generation of soluble Trem2 similar to patients with the homozygous p.T66M mutation. Trem2 p.T66M knock‐in mice show delayed resolution of inflammation upon in vivo lipopolysaccharide stimulation and cultured macrophages display significantly reduced phagocytic activity. Immunohistochemistry together with in vivo TSPO small animal positron emission tomography (μPET) demonstrates an age‐dependent reduction in microglial activity. Surprisingly, perfusion magnetic resonance imaging and FDG‐μPET imaging reveal a significant reduction in cerebral blood flow and brain glucose metabolism. Thus, we demonstrate that a TREM2 loss‐of‐function mutation causes brain‐wide metabolic alterations pointing toward a possible function of microglia in regulating brain glucose metabolism.

Creation of targeted genomic deletions using TALEN or CRISPR/Cas nuclease pairs in one‐cell mouse embryos

The use of TALEN and CRISPR/CAS nucleases is becoming increasingly popular as a means to edit single target sites in one‐cell mouse embryos. Nevertheless, an area that has received less attention concerns the engineering of structural genome variants and the necessary religation of two distant double‐strand breaks. Herein, we applied pairs of TALEN or sgRNAs and Cas9 to create deletions in theRab38 gene. We found that the deletion of 3.2 or 9.3 kb, but not of 30 kb, occurs at a frequency of 6–37%. This is sufficient for the direct production of mutants by embryo microinjection. Therefore, deletions up to ∼10 kb can be readily achieved for modeling human disease alleles. This work represents an important step towards the establishment of new protocols that support the ligation of remote DSB ends to achieve even larger rearrangements.

MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults

MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype. Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as a new valuable approach, allowing it to discriminate between gene function in the adult and the developing postnatal brain.

Generation of targeted mouse mutants by embryo microinjection of TALENs

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.

Control of gene editing by manipulation of DNA repair mechanisms

DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

Gene editing in mouse zygotes using the CRISPR/Cas9 system.

The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.