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The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease., (© 2024. The Author(s).)

Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.

Knockout mice are used extensively to explore the phenotypic effects of mammalian gene dysfunction. With the application of RNA-guided Cas9 nuclease technology for the production of knockout mouse lines, the time, as well as the resources needed, to progress from identification of a gene of interest to production of a knockout line is significantly reduced. Here we present our standard methodology to produce knockout mouse lines by the electroporation of Cas9 ribonucleoprotein (RNP) into mouse zygotes. Using this protocol, we have obtained an 80% success rate in the generation of founders for null alleles with a subsequent 93% germline transmission rate. These methods rely on equipment already present in the majority of transgenic facilities and should be straightforward to implement where appropriate embryo handling expertise exists., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G 2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos., (© 2020 John Wiley & Sons, Inc.)

Large fragment knock-in mouse models such as reporters and conditional mutant mice are important models for biological research. Here we describe 2-cell (2C)-homologous recombination (HR)-CRISPR, a highly efficient method to generate large fragment knock-in mouse models by CRISPR-based genome engineering. Using this method, knock-in founders can be generated routinely in a time frame of about two months with high germline transmission efficiency. 2C-HR-CRISPR will significantly promote the advancement of basic and translational research using genetic mouse models.

Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.

Frequently, there is a need for fluorescent protein detection in mouse cell cultures, including embryonic stem cells or their differentiated derivatives, primary and transformed cells. Here, cells expressing green fluorescent protein-labeled proteins are observed using fluorescent microscopy., (© 2019 Cold Spring Harbor Laboratory Press.)

Fluorescent proteins (FPs) are popular reporters available for gene expression detection and determination of cellular identities in the mouse. This protocol can be used to detect green fluorescent protein spectral variants and proteins labeled with the fusion tag dsRed in fixed cells., (© 2019 Cold Spring Harbor Laboratory Press.)

DNA samples are prepared from mouse tail tips by Proteinase K digestion and subsequently extracted. The resulting preparation is suitable for use in Southern blotting or polymerase chain reaction (PCR)., (© 2019 Cold Spring Harbor Laboratory Press.)

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). In this protocol, a small piece from the tip of the tail is removed and processed using hot sodium hydroxide and Tris (HotSHOT)., (© 2019 Cold Spring Harbor Laboratory Press.)

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Small tissues of a few hundred cells are used for genotyping early postimplantation- and preimplantation-stage embryos by polymerase chain reaction (PCR)., (© 2019 Cold Spring Harbor Laboratory Press.)

To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the tet-O promoter. Therefore, the challenge of getting this system working properly is a serious prospect. In this protocol, we describe how to identify a silent but highly activatable genomic site by taking advantage of transgenic lines reliably expressing the tetracycline transactivators from the Rosa-26 locus. These lines provide optimal Tet-inducible expression: There is minimal leakiness at the "off" state and a high level of induction in the presence of the inducer, doxycycline. The procedure requires (1) an embryonic stem (ES) cell line (germline competent) expressing rtTA from the Rosa-26 locus and (2) construction of a Tet-inducible transgene. The transgene contains the tet-O promoter followed by the gene of interest linked to a βgeo gene (a fusion between lacZ and neo ) through an internal ribosomal entry site (IRES) sequence, which allows the initiation of translation in a cap-independent manner., (© 2018 Cold Spring Harbor Laboratory Press.)

The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2., Competing Interests: The authors have declared that no competing interests exist.

Mice carrying patient-associated point mutations are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence-the orthologous sequence for point mutations or the N or C terminus for epitope tags-appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening of born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target mutations introduced by Cas9. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Fluorescent protein (FP)-expressing transgenic mice are powerful genetic resources for marking both live and fixed cells and tissues. Expression in live embryos requires only dissection and visualization using an appropriate microscope. Fixation can compromise FP activity. A simple tissue-clearing agent called Sca l e preserves FP activity while rendering the embryo or organ optically transparent., (© 2018 Cold Spring Harbor Laboratory Press.)

For experiments that require large numbers of preimplantation mouse embryos, such as microinjection of zygotes, gonadotropins are administered to females before mating to increase the number of oocytes that are ovulated (i.e., to induce superovulation). Pregnant mare serum gonadotropin (PMSG) is used to mimic the oocyte maturation effect of the endogenous follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) is used to mimic the ovulation induction effect of luteinizing hormone (LH)., (© 2018 Cold Spring Harbor Laboratory Press.)

The variability in embryonic stem (ES) cell culture is due primarily to serum. Serum is typically produced in large batches from many animals. However, samples may differ depending on the age and diet of the animals, the country of origin, and other factors creating lot-to-lot variations. Some vendors test FBS lots for compatibility with ES cell culture. Many laboratories prefer to test serum batches themselves to identify the lot giving optimal growth. In this protocol, small quantities of specific serum batches are obtained from different suppliers and tested for their ability to support ES cells in an undifferentiated state. A complete test includes the serum batches' influence on plating efficiency, cell morphology, toxicity, and, if possible, their ability to support generation of chimeras., (© 2017 Cold Spring Harbor Laboratory Press.)

Characterization of embryonic stem (ES) cell-mediated genome alterations, including random insertional transgenesis, gene trapping, gene targeting, and site-specific recombinase-mediated changes, is performed mostly at the ES cell level, before the introduction of these alterations into a mouse. A detailed characterization requires a larger amount of DNA than is required for the initial detection of the candidates for the desired alteration. This protocol describes the preparation of DNA from a 10-cm tissue culture plate. The cells are trypsinized and lysed, and DNA is recovered from the lysate by organic extraction followed by ethanol precipitation., (© 2017 Cold Spring Harbor Laboratory Press.)

Mouse embryonic fibroblasts can be reprogrammed to ES cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The piggyBac transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. We found that FuGENE HD transfection reagent can be effective for mouse embryonic fibroblasts (MEFs) to generate induced pluripotent stem cells (iPSCs) with the piggyBac transposon transgenes. There are multitudes of cell transfection methods commercially available. Their efficiency, and thus their success, in inducing pluripotent cell types are cell-type-dependent., (© 2017 Cold Spring Harbor Laboratory Press.)

In 2006, Shinya Yamanaka and his student Kazutoshi Takahashi showed that the expression of only four specific genes is sufficient to reprogram fully differentiated somatic cells into pluripotent stem cells. These cells, termed induced pluripotent stem (iPS) cells, share many of their characteristics with embryonic stem (ES) cells. In this protocol, we describe one of the simplest ways of generating iPS cells from mouse fibroblasts. It combines an efficient transposon-mediated transfection and the tetracycline-inducible system to control the expression of the Yamanaka reprogramming factors., (© 2017 Cold Spring Harbor Laboratory Press.)

Mouse embryonic fibroblasts can be reprogrammed to embryonic stem (ES) cell-like pluripotent stem cells by the forced expression of four transcription factors-OCT4, SOX2, KLF4, and c-MYC. The piggyBac transposon system has proven effective as a vehicle for the delivery of transgenes into fibroblasts and for successful reprogramming to induced pluripotent stem (iPS) cells. This protocol is designed for use with the Neon electroporation system. It can be adapted to other types of electroporation systems., (© 2017 Cold Spring Harbor Laboratory Press.)

Sharing genetically modified mouse models is a very important part of collaboration between researchers. Shipping live animals around the world is inconvenient, expensive, and cumbersome because of the variety of international regulations and paperwork. The issue of health status differences between animal facilities is of great importance; traditionally, imported animals are quarantined to determine their health status and avoid the introduction of undesirable pathogens. The shipment of preimplantation-stage embryos for immediate transfer into pseudopregnant recipients upon arrival is a commonly used method for transportation. Time coordination on both sides is critical in this case, but the shipment can be done by any courier and the container does not need to be returned. This protocol has been used since the early 1990s to rederive dozens of mouse strains., (© 2017 Cold Spring Harbor Laboratory Press.)

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size., (© 2016 Cold Spring Harbor Laboratory Press.)

Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.

The female mouse estrous cycle is divided into four phases: proestrus (development of ovarian follicles), estrus (ovulation), metestrus (formation of corpora lutea), and diestrus (beginning of follicle development for next ovulation and elimination of previous oocytes). The appearance of the epithelium of the external genitalia is used to identify the stage of the estrous cycle of a female mouse. This is usually easier to see in strains with either no or only light skin pigmentation. By examining the color, moistness, and degree of swelling of the vagina, females in estrus can readily be identified. To set up the matings, females are examined in the afternoon, and those in estrus are placed into the cages with males (one or two females in each cage with one male). Usually, 50% or more of the selected females will mate. The presence of a vaginal copulation plug next morning indicates that mating has occurred, but it does not mean that a pregnancy will result even if proven breeder fertile males were used. It is important to check vaginal plugs early in the morning because they fall out or are no longer detectable ~12 h after mating or sometimes earlier., (© 2016 Cold Spring Harbor Laboratory Press.)

Production of the germline-competent chimeras using genetically modified ES cell lines is an essential step in the establishment of novel mouse models. In addition chimeras provide a powerful tool to study the cell lineage and to analyze complex phenotypes of mutant mice. Mouse chimeras with tetraploid embryos are used to rescue extraembryonic defects, to analyze an impact of gene function on specific lineage, to study the interaction between embryonic and extraembryonic tissues, and to produce mutant embryos and mice for the phenotype analysis. Tetraploid embryos are generated by the fusion of two blastomeres of the mouse embryo. The applications of tetraploid complementation assay and the protocol are described below.

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.