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Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genetics approaches have been instrumental to deciphering T cell development in the thymus, including gene disruption by homologous recombination and more recently Crispr-based gene editing and transgenic gene expression, especially of specific T cell antigen receptors (TCR). This brief chapter describes commonly used tools and strategies to modify the genome of thymocytes, including mouse strains with lineage- and stage-specific expression of the Cre recombinase used for conditional allele inactivation or expressing unique antigen receptor specificities., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Advances in CRISPR/Cas9 genome editing technologies have allowed for the rapid generation of Cre-loxP conditional knockout mice. However, current strategies for genotyping flox mice, typically based on Sanger sequencing following cloning of target sequences from dozens of pups, are time-consuming. Here, we describe a rapid screening method for flox mice, using in vitro Cre recombination that can be performed using simple enzymatic reactions and enables detection of functional flox mouse within 1 day. In addition, we introduce an efficient strategy for subsequent sequence analysis by cloning of floxed regions using the In-Fusion system. Our genotyping pipeline reduces laborious tasks and thus contributes to the rapid selection of accurately edited flox mice., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes.Here we report a protocol for the generation of targeted transgenic alleles at Gt(ROSA)26Sor, taking as an example a conditional overexpression allele, by PhiC31 integrase/recombinase-mediated cassette exchange of an engineered Gt(ROSA)26Sor locus in mouse embryonic stem cells. The overexpression allele is initially silenced by the presence of a loxP flanked stop sequence but can be strongly activated through the action of Cre recombinase., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genetic lineage tracing is accomplished using bi-transgenic mice, where one allele is altered to express Cre recombinase, and another allele encodes a Cre-dependent genetic reporter protein. Once Cre is activated (constitutive or in response to tamoxifen), the marker gene-expressing cells become indelibly labeled by the reporter protein. Therefore, daughter cells derived from labeled cells are permanently labeled even if the marker gene that drove Cre recombinase expression is no longer expressed in these cells. This system is commonly used to label putative progenitor cells and determine the fate of their progeny. Here, we describe the use of c-kit-based genetic lineage-tracing mouse line as an example and discuss caveats for performing these types of experiments.

Cartilage is a specialized skeletal tissue with a unique extracellular matrix elaborated by its resident cells, chondrocytes. The tissue presents in several forms, including growth plate and articular cartilage, wherein chondrocytes follow a differential differentiation program and have different fates. The induction of gene modifications in cartilage specifically relies on mouse transgenes and knockin alleles taking advantages of transcriptional elements primarily active in chondrocytes at a specific differentiation stage or in a specific cartilage type. These transgenes/alleles have been widely used to study the roles of specific genes in cartilage development, adult homeostasis, and pathology. As cartilage formation is critical for postnatal life, the inactivation or significant alteration of key cartilaginous genes is often neonatally lethal and therefore hampers postnatal studies. Gold standard approaches to induce postnatal chondrocyte-specific gene modifications include the Cre-loxP and Tet-ON/OFF systems. Selecting the appropriate promoter/enhancer sequences to drive Cre expression is of crucial importance and determines the specificity of conditional gain- or loss-of-function models. In this chapter, we discuss a series of transgenes and knockin alleles that have been developed for gene manipulation in cartilage and we compare their expression patterns and efficiencies.

Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the bar au gene flanked by loxP sites in the plastid genome. For marker excision Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar au gene, confers a visual aurea leaf phenotype, thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The success of in planta plastid marker excision proves that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA and the availability of visual marker genes.

Minicircle DNA (mcDNA) has been considered to be an alternative choice of traditional DNA vaccine due to its much smaller size, resulting in more efficient antigen synthesis, enhanced and long-lasting adaptive immune response, especially cellular immune response. However, the disadvantages such as relative high cost and labor intensiveness severely restrict its direct application in the field of veterinary vaccine. Here, we describe a novel Cre Recombinase-mediated In vivo McDNA platform, named CRIM, in which the parental plasmid could spontaneously transform into mcDNA by itself after transfection or oral administration. This CRIM vaccine platform might serve as a novel oral antigen delivery system for any infectious diseases, especially for veterinary application.

Cre-mediated recombination has become a powerful tool to confine gene deletions (conditional knockouts) or overexpression of genes (conditional knockin/overexpression). By spatiotemporal restriction of genetic manipulations, major problems of classical knockouts such as embryonic lethality or pleiotropy can be circumvented. Furthermore, Cre-mediated recombination has broad applications in the analysis of the cellular behavior of subpopulations and cell types as well as for genetic fate mapping. This chapter gives an overview about applications for the Cre/LoxP system and their execution.

Intestinal stem cells are responsible for tissue renewal. The study of stem cell properties has become a major challenge in the field. We describe here a method based on Cre recombinase inducible lentivirus vectors that permits delivery of transgenes, either for overexpression or knockdown, in primary stem cells that can be cultured in an 3D intestinal organoid system. This method is an excellent approach for genetic manipulation and can complement in vivo transgenic experiments.

The use of inducible transgenic Nestin-CreER T2 mice has proved to be an essential research tool for gene targeting and studying the molecular pathways implicated in adult neurogenesis, namely, inside the adult subgranular zone (SGZ) of the dentate gyrus and the adult subventricular zone (SVZ) lining the lateral ventricles. Several lines of Nestin-CreER-expressing mice were generated and used in adult neurogenesis research in the past two decades; however, their suitability for studying neurogenesis in aged mice remains elusive. Here, we assessed the efficiency of Cre-loxP genetic recombination in the aging SVZ using the Nestin-CreER T2 /Rosa26YFP line designed by Lagace et al. (J Neurosci 27(46):12623-12629, 2007). This analysis was performed in 12-month-old (middle-aged) mice and 20-month-old (old) mice compared to 2-month-old (young adult) mice. To evaluate successful recombination, our approach relies on the histological assessment of Cre mRNA level of expression and the YFP reporter gene's expression inside the aging SVZ by combining in situ hybridization and immunohistochemistry. Using co-immunolabeling, this approach also provides the advantage of estimating the percentage of recombined progeny [(GFP+Nestin+)/Nestin+] and the rate of cell proliferation [(GFP+Ki67+)/GFP+] inside the aging SVZ niche.

Over the last several decades, multiple recurrent chromosomal amplifications and deletions have been detected in a large number of cancers. These regions of amplification and deletion can encompass a few to several hundred genes. Determining which of these genes is causing the outgrowth of the cancer is difficult. Complicating the analysis is the fact that several genes within the affected chromosomal region may cooperate to promote tumorigenesis. In this protocol we describe a method of chromosomal engineering in mice that allows modeling of chromosomal duplications and deficiencies. This method faithfully recapitulates several aspects of chromosomal loss and gain in human cancers and can reveal cancer drivers difficult to identify by other means.

The discovery of new gene editing tools in the past several years has moved the transgenic field to a new level. The traditional random transgenesis method by pronuclear microinjection has been largely replaced by targeted or site-specific transgenic technologies without the need of homologous recombination in embryonic stem (ES) cells. In this chapter, I describe detailed protocols of an integrase-based approach, trademarked as "TARGATT™" (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into a predetermined chromosomal locus with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3 months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Biotechnology methods for inserting genes one by one or as a block of fragment into plant genomes are needed to introduce valuable traits into crop varieties. Insertion of multiple genes into a single site, called as molecular stacking, is important to allow co-inheritance of the genes into the progeny. Generally, two approaches are available for creating gene stacks: nuclease-induced targeted gene integration into native sites and recombinase-mediated gene integration into the engineered sites. The recombinase application is attractive as several recombinases show high efficiency and precision in plant genomes. This chapter describes a gene stacking method based on the use of Cre-lox site-specific recombination system to integrate genes into the engineered sites and nucleases to delete selection genes leading to stacking of traits into a single genomic site. High efficiency and precision, and undetectable off-target effects of Cre-lox in a number of plant species, make it an attractive tool for complex applications such as gene stacking.

For studies of gene function during development, it can be very useful to generate mosaic embryos in which a small subset of cells in a given cell lineage lacks a gene of interest and carries a marker that allows the mutant cells to be specifically visualized and compared to wild-type cells. Several methods have been used to generate genetically mosaic mouse kidneys for such studies. These include (1) chimeric embryos generated using embryonic stem cells, (2) chimeric renal organoids generated by dissociation and reaggregation of the fetal kidneys, (3) generation of a knockout allele with a built-in reporter gene, (4) mosaic analysis with double markers (MADM), and (5) mosaic mutant analysis with spatial and temporal control of recombination (MASTR). In this chapter, these five methods are described, and their advantages and disadvantages are discussed.

Cell-type-specific gene targeting with the Cre/loxP system has become an indispensable technique in experimental neuroscience, particularly for the study of late-born glial cells that make myelin. A plethora of conditional mutants and Cre-expressing mouse lines is now available to the research community that allows laboratories to readily engage in in vivo analyses of oligodendrocytes and their precursor cells. This chapter summarizes concepts and strategies in targeting myelinating glial cells in mice for mutagenesis or imaging, and provides an overview of the most important Cre driver lines successfully used in this rapidly growing field.

The discovery of Lgr5 as a marker of adult stem cells meant that stem cell populations could be purified and studied in isolation. Importantly, when cultured under the appropriate conditions these stem cells form organoids in tissue culture that retain many features of the tissue of origin. The organoid cultures are accessible to genetic and biochemical manipulation, bridging the gap between in vivo mouse models and conventional tissue culture. Here we describe robust protocols to establish organoids from gastrointestinal tissues (stomach, intestine, liver) and Cre-recombinase mediated gene manipulation in vitro.

Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a Gαs-coupled DREADD (GsD) fused to GFP in distinct cell populations in vivo. These animals also harbor a CREB-activated luciferase reporter gene for analysis of CREB activity by in vivo imaging, ex vivo imaging, or biochemical reporter assays. In this chapter, we provide detailed methods for breeding GsD animals, inducing GsD expression, stimulating GsD activity, and measuring basal and stimulated CREB reporter bioluminescence in tissues in vivo, ex vivo, and in vitro. These animals are available from our laboratory for non-profit research.

Myelination in the central nervous system takes place predominantly during the postnatal development of humans and rodents by myelinating oligodendrocytes (OLs), which are differentiated from oligodendrocyte progenitor cells (OPCs). We recently reported that Sox2 is essential for developmental myelination in the murine brain and spinal cord. It is still controversial regarding the role of Sox2 in oligodendroglial lineage progression in the postnatal murine spinal cord. Analyses of a series of cell- and stage-specific Sox2 mutants reveal that Sox2 plays a biphasic role in regulating oligodendroglial lineage progression in the postnatal murine spinal cord. Sox2 controls the number of OPCs for subsequent differentiation through regulating their proliferation. In addition, Sox2 regulates the timing of OL differentiation and modulates the rate of oligodendrogenesis. Our experimental data prove that Sox2 is an intrinsic positive timer of oligodendroglial lineage progression and suggest that interventions affecting oligodendroglial Sox2 expression may be therapeutic for overcoming OPC differentiation arrest in dysmyelinating and demyelinating disorders.

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7 f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7 f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7 f/f Tyr-Cre mice relative to Atg7 f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7 f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7 f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1 Cre mice with Exoc5 f/f mice for hair cell-specific deletion (Gfi1 Cre/+ ;Exoc5 f/f ) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5 f/f ). Gfi1 Cre/+ ;Exoc5 f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5 f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.

Methylobacterium extorquens AM1 is the model strain for methylotrophic bacteria that metabolize methanol as the sole carbon and energy source. Genetically modified M. extorquens AM1 is used as a methylotrophic cell factory (MeCF) for high value-added chemical production. We tested the Cre-loxP recombination system for its ability to mediate multicopy gene integration of the mvt3 operon (mvt3) in M. extorquens AM1. mvt3 controls the expression of the first three enzymes of the mevalonate synthesis pathway. We assayed for Cre-mediated multigene integration by screening for multicopy mutants via their survival in culture with a high kanamycin concentration (600 μg/mL). We identified mutant strains in which the mevalonate titer was increased by up to 1.9-fold compared with M2 (M. extorquens AM1ΔcelABCΔattTn7::mvt3::loxP) and confirmed mvt3 integration at 2-3 copies per genome. This result demonstrates the feasibility of multicopy integration in M. extorquens AM1 mediated by Cre-loxP recombination and its potential for improving the output of M. extorquens AM1 metabolic pathways, e.g., optimization of terpenoid synthesis.

The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from DNA replication until anaphase. In mammals, meiotic DNA replication occurs in oogonia of embryos and chromosome segregation occurs in oocytes of sexually mature females. Sister chromatid cohesion establishment and chromosome segregation are thus separated by months in the mouse and decades in the human. The meiotic cohesin complex that maintains sister chromatid cohesion must therefore hold replicated sisters together for a long time in oocytes. Remarkably, this is achieved by establishing cohesion exclusively in prenatal oocytes. Meiotic cohesion in females is maintained without detectable turnover and cohesin is therefore thought to be a long-lived protein complex. Nevertheless, the lifespan of cohesin molecules is limited as chromosomal cohesin levels decline with maternal age. The age-related loss of cohesin and weakened cohesion correlate with an age-related increase in chromosome missegregation of meiosis I oocytes that results in aneuploid eggs. Therefore, loss of chromosomal cohesin has been proposed to be a leading cause of the maternal age effect. To better understand cohesin deterioration in oocytes, it is crucial to gain insights into mammalian cohesion establishment and maintenance mechanisms by manipulating cohesin in live oocytes.This chapter describes techniques that address the manipulation of meiotic cohesin levels in mouse oocytes. First, we describe how cohesin can be efficiently removed from meiotic chromosomes by injecting mRNA encoding TEV protease in live oocytes expressing cohesin with engineered TEV recognition sites, followed by imaging. Secondly, we describe how cohesin expression can be induced during different stages of oocyte development using genetically modified mouse strains. In particular, we describe how to determine the deletion timing of germline-specific Cre recombinases using β-galactosidase staining of fetal ovaries. Lastly, we provide guidance on how to quantify cohesin levels on metaphase I chromosome spreads.

Lineage tracing allows for identification of all progeny produced by a single cell or groups of cells and can thus be used to assess developmental fate of cells. Here we focus on one of the most widely used lineage tracing approaches that utilize the Cre/loxP system for site-specific genetic recombination in studying the developmental origins of lymphatic endothelial cells (LECs) in the mouse embryo. We discuss general considerations for a successful Cre/loxP based lineage tracing experiment and provide information about strains that are available for genetic lineage tracing of LECs. A protocol for lineage tracing analysis of the lymphatic vasculature by whole-mount immunofluorescence in two embryonic tissues, the skin and the mesentery, is also provided.

The bacteriophage P1 Cre/lox system has been utilized in diverse fungi for marker recycling and exchange, generation of targeted chromosome translocations, and targeted deletion of interstitial chromosome segments. Here we show the application of this tool in the wheat and maize pathogen, Fusarium graminearum. We explored three different ways to introduce Cre into strains with floxed genes, namely transformation with an episomal or integrative plasmid (pLC28), fusion of protoplasts of strains carrying floxed genes with strains expressing Cre by forcing heterokaryons, and crosses between strains with floxed genes and strains expressing Cre to isolate progeny in which the target genes had been deleted during the cross. We used this system for the construction of strains bearing auxotrophic markers that were generated by gene replacement with positively selectable markers followed by Cre-mediated marker excision. In addition, updated protocols for transformation and crosses for F. graminearum are provided. In combination, strains and tools developed here add to the arsenal of methods that can be used to carry out molecular genetics with F. graminearum.

Transgenic mice have emerged as a central tool in the study of lung myeloid cells during homeostasis and disease. The use of Cre/Lox site-specific recombination allows for conditional deletion of a gene of interest in a spatially controlled manner. The basic Cre/Lox system can be further refined to include an inducible trigger, enabling conditional deletion of a gene of interest in a spatially and temporally controlled manner. Here we provide an overview of commercially available conditional and inducible conditional mouse strains that target lung myeloid cells and describe the appropriate breeding schemes and controls for transgenic animal systems that can be used to modulate myeloid cell function.

Lung cancers display considerable intertumoral heterogeneity, leading to the classification of distinct tumor subtypes. Our understanding of the genetic aberrations that underlie tumor subtypes has been greatly enhanced by recent genomic sequencing studies and state-of-the-art gene targeting technologies, highlighting evidence that distinct lung cancer subtypes may be derived from different "cells-of-origin". Here, we describe the intra-tracheal delivery of cell type-restricted Ad5-Cre viruses into the lungs of adult mice, combined with immunohistochemical and flow cytometry strategies for the detection of lung cancer-initiating cells in vivo.

The BAF chromatin remodeling complex plays an essential role in brain development. However its function in postnatal neurogenesis in hippocampus is still unknown. Here, we show that in postnatal dentate gyrus (DG), the BAF170 subunit of the complex is expressed in radial glial-like (RGL) progenitors and in cell types involved in subsequent steps of adult neurogenesis including mature astrocytes. Conditional deletion of BAF170 during cortical late neurogenesis as well as during adult brain neurogenesis depletes the pool of RGL cells in DG, and promotes terminal astrocyte differentiation. These derangements are accompanied by distinct behavioral deficits, as reflected by an impaired accuracy of place responding in the Morris water maze test, during both hidden platform as well as reversal learning. Inducible deletion of BAF170 in DG during adult brain neurogenesis resulted in mild spatial learning deficits, having a more pronounced effect on spatial learning during the reversal test. These findings demonstrate involvement of BAF170-dependent chromatin remodeling in hippocampal neurogenesis and cognition and suggest a specific role of adult neurogenesis in DG in adaptive behavior.

MicroRNA-8 and its target gene, u-shaped (ush), regulate body size in Drosophila. As the mouse homolog of the Drosophila miR-8, whether the miR-200 family has similar functions and how they perform their regulatory roles in body size control is unknown. In order to discover the biological function of the miR-200s in vivo, we generated mice lacking the miR-200b/200a/429 gene cluster using the Cre/loxp system. miR-200b/200a/429 null mutant mice were viable, fertile, and showed normal development.

Large insert mate pair reads have been used in de novo assembly and discovery of structural variants. We developed a new approach, Cre-LoxP inverse PCR paired end (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends. We have successfully created CLIP-PE libraries of up to 22 kb jumping pairs and demonstrated their ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms.

The protein sequences of class I HDACs in mice and humans are 96-99 % identical. These highly conserved proteins have crucial roles in biological processes, such as proliferation and development, which is reflected in the lethality that occurs in conventional whole body knockout mice. Therefore, conditional knockouts are inevitable to investigate the functions of class I HDACs in mice. Here, we describe the generation of conditional class I Hdac knockout mice, using Hdac1 as an example. We explain a relatively quick procedure to generate the necessary target vectors by recombination-mediated genetic engineering and gateway techniques. Furthermore, we show how to culture, target, and screen for positively recombined ES cells. Additionally, we present a dual recombination system, which allows the deletion of class I Hdacs at any time by a tamoxifen inducible Cre.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ϕC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies.

Expression of activation-induced cytidine deaminase (AID) is the hallmark of B cells engaged in an immune response in germinal centers. We designed an inducible fate-mapping reporter mouse in which AID-expressing B cells could be timely and irreversibly marked, by knockin at the Aicda locus of a tamoxifen-inducible Cre recombinase. This mouse model allows notably for the long-term follow-up of memory B cells and plasma cells engaged in an immune response. We describe here a protocol to generate hybridomas from small memory subsets that can be easily traced and identified in this mouse line through Cre-activated fluorescent reporters.

The germinal center (GC) reaction represents an essential phase of an adaptive immune response. Dysfunction of GC B cells can lead to life-threatening diseases including autoimmune disorders, lymphomas, and opportunistic infections. Defining the molecular circuitries controlling GC B cell physiology is crucial to understand the pathogenesis of GC B cell disorders, as well as to develop improved vaccines against foreign pathogens. Conditional gene targeting based on the Cre/loxP recombination system has substantially accelerated our comprehension of the genetic networks controlling GC B cell function. Several independent studies in the past 10 years have highlighted the many advantages and the few limitations and pitfalls associated to conditional gene manipulation in GC B cells using the Cre/loxP recombination system. Here, we describe the basic features of GC B cell-specific gene targeting experiments. We provide indications on the type of Cre transgene and controls to be chosen, way-out strategies to overcome leakiness of the Cre/loxP system, and approaches to minimize the number of experimental animals and to speed up analyses on conditional mutant GC B cells.

The technique of Cre-mediated DNA recombination at loxP sites has been used widely in manipulation of the genome in cultured cells and in living organisms. Local delivery of Cre recombinase protein tagged with a cell-penetrating (or permeable) peptide (Cre-CPP) has the advantage of additional spatial and temporal control when compared to genetic delivery methods. In this chapter, we describe protocols for injection-based intramuscular delivery of Cre-CPP dissolved in hydrogel to skeletal muscle and by ultrasound-guided injection to cardiac muscle in mice.

Delivery of proteins to the cytosol of living cells is a promising research tool. Delivery of antibodies in particular bears exciting applications such as in vivo tracking of proteins at endogenous expression levels or interference with cellular processes. In spite of the large number of methods published for protein delivery, successful applications so far are rare. A possible explanation for this is a vast overestimation of the delivery efficiency due to the use of inappropriate detection methods and/or unsuitable positive controls for cytosolic delivery. Therefore, we provide strategies for unequivocally detecting cytoplasmic protein delivery and quantifying protein transformation efficiency. Finally, we present a protocol for efficient protein delivery to the cytosol validated using these methods.

When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone. When a single recombinase is used for RMCE, recombination between undesired pairings of the sites can lead to a significant number of unwanted cell lines. To reduce the frequency with which these side products occur, several variants of RMCE that increase desired outcomes have been developed. Nevertheless, an important feature lacking from these improved RMCE methods is that none have fully utilized the recombinases that have been demonstrated to be the most robust and stringent at performing genomic integration in plants and animals, i.e., the phiC31 and Bxb1 phage integrases. To address this need, we have developed an RMCE protocol using these two serine integrases that we call dual integrase cassette exchange (DICE). Our DICE system provides a means to achieve high-precision DNA integration at a desired location and is especially well suited for repeated recombination into the same locus. In this chapter, we provide our most current protocols for using DICE in feeder-free human-induced pluripotent stem cells .

Fosmid end sequencing has been widely utilized in genome sequence assemblies and genome structural variation studies. We have developed a new approach to construct fosmid paired-end libraries that is suitable for Illumina sequencing platform. This approach employs a newly modified fosmid vector (pFosClip) which contains two loxP sites with identical orientation and two inverse Illumina adaptor priming sites flanking the cloning site. DNA prepared from the fosmid library constructed with pFosClip can be treated with the Cre recombinase to remove most of the vector DNA, leaving only 107 bp of the vector sequence with insert DNA. Frequent cutting restriction enzymes and ligase are used to digest the fosmid DNA to small (less than 1 Kb) fragments and recircularize the fosmid ends and all the internal fragments. Finally an inverse PCR step with the Illumina primers is used to enrich the fosmid paired ends (PEs) for sequencing. The advantages of this approach are the following: (1) the circularization of short fragments with sticky ends is efficient; therefore the success rate is higher than other approaches that attempt to join both blunt ends of large fosmid vectors; and (2) the restriction enzyme cutting generates an identifiable junction tag for splitting the paired reads. (3) Multiple restriction enzymes can be used to overcome possible enzyme-cutting bias. Our results have shown that this approach has produced mostly fosmid size (30-40 Kb) pairs from the targeted fungi and plant genomes and has drastically increased the scaffold sizes in the assembled genomes.

The fluorescent protein revolution has made the light microscope the most widely used tool for studying biological structure from the single-molecule to whole organism scales. However, traditional approaches are limited in their ability to resolve components in highly complex structures, such as the brain. In recent years, this limitation has been circumvented by the development of multicolor labeling methods, termed Brainbow. Brainbow tools rely on site-specific recombinases to make stochastic "choices" between different combinations of fluorescent proteins so that structures in close proximity to one another can be resolved based on their color profile. These new approaches, however, call for more refined methods of sample preparation and imaging optimized for multispectral imaging, which are presented here. The most robust approach for generating useful Brainbow data combines immunohistology with multispectral laser scanning confocal microscopy. This chapter, therefore, focuses on this particular technique, though the imaging principle discussed here is applicable to other Brainbow approaches as well.

In this chapter we present an alternative method to develop marker-free transgenic plants. It makes use of the Cre/loxP recombination system from bacteriophage P1 and consists of two essential components. The first component is the transgenic plant containing a loxP-flanked marker gene. The second component is a cre transient expression vector based on potato virus X. The great benefit of this transient delivery method consists in the avoidance of stable integration of the cre recombinase gene into the plant genome. Upon infection of the loxP-target plant with PVX-Cre, the virus spreads systemically through the plant and causes the recombinase-mediated excision of the marker gene. Marker-free transgenic loci can be transmitted to the progeny by plant regeneration from PVX-Cre systemically infected leaves or self-pollination of virus-infected plants. The protocol covers generation of loxP-target transgenic plants, PVX-mediated delivery of Cre recombinase protein, phenotypic and molecular analysis of recombination events, and transmission of marker-free transgenic loci to the next generation. The transient expression system described in this chapter can be adapted for marker gene removal in other plant species that are amenable for virus infection.

Histone deacetylases (HDACs) play crucial roles during mammalian development and for cellular homeostasis. In addition, these enzymes are promising targets for small molecule inhibitors in the treatment of cancer and neurological diseases. Conditional HDAC knock-out mice are excellent tools for defining the functions of individual HDACs in vivo and for identifying the molecular targets of HDAC inhibitors in disease. Here, we describe the generation of tissue-specific HDAC knock-out mice and delineate a strategy for the generation of conditional HDAC knock-in mice.

Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology. This one-step generation of mice is easier in design and faster. Here, we describe our achieved success rate, the considerations in design of a conditional allele, a detailed protocol to prepare the zygote injection mix, and the screening procedure to identify the new conditional knockout mouse strain.

Obesity and Type 2 diabetes (T2D) are associated with a variety of comorbidities that contribute to mortality around the world. Although significant effort has been expended in understanding mechanisms that mitigate the consequences of this epidemic, the field has experienced limited success thus far. The potential ability of brown adipose tissue (BAT) to counteract obesity and metabolic disease in rodents (and potentially in humans) has been a topical realization. Recently, there is also another thermogenic fat cell called beige adipocytes, which are located among white adipocytes and share similar activated responses to cyclic AMP as classical BAT. In this chapter, we review contemporary molecular strategies to investigate the role of adipose tissue depots in metabolism. In particular, we will discuss the generation of adipose tissue-specific knockout and overexpression of target genes in various mouse models. We will also discuss how to use different Cre (cyclization recombination) mouse lines to investigate diverse types of adipocytes.

Recombinase-mediated cassette exchange, or RMCE, is a genome engineering tool that can be used to swap DNA fragments of interest between two DNA molecules. In a variation of RMCE, called dual RMCE, the exchange of DNA fragments is mediated by two recombinases in contrast to one recombinase in the classic RMCE reaction. Under optimal conditions, the efficiency of dual RMCE can be quite high: up to ~45% of the transfected cells depending on the recombinase pair used to mediate the replacement reaction. Here we describe protocols for preparing for, performing, and optimizing the parameters of dual RMCE.

We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants. Using the experimental procedures and conditions described here, as high as 20% of bombarded embryos can produce regenerable recombinant callus events. This nanomaterial-mediated, DNA-free methodology has potential to become an effective tool for plant genome editing.

For the genetic alterations of NMDA receptor (NMDAR) properties like Ca 2+ -permeability or voltage-dependent gating in mice and for the experimental analysis of nonsense or missense mutations that were identified in human patients, single nucleotide mutations have to be introduced into the germ line of mice (Burnashev and Szepetowski, Curr Opin Pharmacol 20:73-82, 2015; Endele et al., Nat Genet 42:1021-1026, 2010). This can be done with very high precision by the well-established method of gene replacement, which makes use of homologous recombination in pluripotent embryonic stem (ES) cells of mice. The homologous recombination at NMDAR subunit genes (Grin; for glutamate receptor ionotropic NMDAR subtype) has to be performed by targeting vectors, also called replacement vectors. The targeting vector should encode part of the gene for the NMDAR subunit, the NMDAR mutation, and a removable selection maker. In these days, the targeting vector can be precisely designed using DNA sequences from public databases. The assembly of the vector is then done from isogenic NMDAR gene fragments cloned in bacterial artificial chromosomes (BACs) using "high fidelity" long-range PCR reactions. During these PCR reactions, the NMDAR mutations are introduced into the cloned NMDAR gene fragments of the targeting vector. Finally, the targeting vector is used for homologous recombination in mouse ES cells. Positive ES cell clones which have the correct mutation have to be selected and are then used for blastocyst injection to generate chimeric mice that hopefully transmit the Grin gene targeted ES cells to their offspring. In the first offspring generation of the founder (F1), some animals will be heterozygous for the targeted NMDAR gene mutation. In order to regulate the expression of NMDAR mutations, it is important to keep the targeted NMDAR mutation under conditional control. Here, we describe a general method how those conditionally controlled NMDAR mutations can be engraved into the germ line of mice as hypomorphic Grin alleles. By breeding these hypomorphic Grin gene targeted mice with Cre recombinase expressing mice, the hypomorphic Grin allele can be activated at specific time points in specific cell types, and the function of the mutated NMDAR can be analyzed in these - so called - conditional mouse models. In this method chapter, we describe in detail the different methodical steps for successful gene targeting and generation of conditional NMDAR mutant mouse lines. Within the last 20 years, several students in our Department of Molecular Neurobiology in Heidelberg used these techniques several times to generate different mouse lines with mutated NMDARs.

The retina is a thin neural tissue sitting on the backside of the eye, composed of light-sensing cells, interneurons, and output ganglion neurons. The latter send electrical signals to higher visual centers in the brain. Transgenic mouse lines are becoming one of the most valuable mammalian animal models for the study of visual signal processing within the retina. Especially, the generation of Cre recombinase transgenic mouse lines provides a powerful tool for genetic manipulation. A key step for the utilization of transgenic lines is the characterization of their transgene expression patterns in the retina. Here we describe a standard protocol for characterizing the expression pattern of the Cre recombinase or fluorescent proteins in the retina with an immunohistochemical approach.

Many genetic tools have been developed that use green fluorescent protein (GFP) and its derivatives for labeling specific cell populations in organisms and in cell culture. To extend the use of GFP beyond labeling purposes, we developed methods and reagents that use GFP as a driver of biological activities. We used nanobodies that bind GFP to engineer CRE-DOG and Flp-DOG, recombinases that can induce Cre/lox and Flp/FRT recombination in a GFP-dependent manner, respectively. Here, we present a protocol to deliver CRE-DOG and Flp-DOG into the mouse brain by recombinant AAV infection. This protocol enables one to manipulate gene expression specifically in GFP-expressing cells, found either in transgenic GFP reporter lines or in cells made to express GFP by other transduction methods.

An inducible tissue-specific knockout (KO) technique has been used to study the role of genes in the adult heart. This KO technique circumvents the developmental effect that could otherwise be observed in a tissue-specific KO. The peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor that when activated has been shown to improve vascular remodeling and endothelial function in hypertensive rodents. Here we describe an inducible tissue specific KO protocol used to study the role of PPARγ in smooth muscle cells (SMC) in angiotensin (Ang) II-induced hypertension in adult mice. Inducible VSMC Pparγ KO mice are generated by crossing mice expressing a fusion protein of Cre recombinase with the modified estrogen receptor ligand binding domain (CreER T2 ) under the control of the smooth muscle myosin heavy chain (smmhc, myh11) with mice having loxP sites flanking exon 2 of the Pparγ gene (Pparγ Flox/Flox ). The SMC Pparγ KO is induced by treating smMHC-CreER T2 /Pparγ Flox/Flox mice with the estrogen receptor antagonist tamoxifen causing recombination of the two loxP site by CreER T2 . SMC KO is confirmed by determining mRNA Pparγ levels in aortic media. Presence of the loxP sites is determined by sequencing genomic DNA. Tissue specific expression is assayed using smMHC-CreER T2 /reporter crossed mice.