In Vivo Optical Interrogation of Neuronal Responses to Genetic, Cell Type-Specific Silencing.

We established a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo Coexpression of a constitutive, Cre-dependent fluorescent marker selectively allowed single-cell analyses before and after inducible, Tet-dependent transgene expression. Here, we used this method for precise, acute manipulation of neuronal activity in the living brain. The goal was to study neuronal network homeostasis at cellular resolution. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least 3 d. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.SIGNIFICANCE STATEMENT Gene function is studied by KO or overexpression of a specific gene followed by analyses of phenotypic changes. However, being able to predict and analyze exactly those cells in which genetic manipulation will occur is not possible. We combined two prominent transgene overexpression methods to fluorescently highlight the targeted cells appropriately before cell type-specific transgene induction. By inducing a potassium channel that decreases neuronal firing, we investigated how neuronal networks in the living mouse brain possibly compensate swift changes in cellular activities. Unlike in vitro, known compensatory homeostatic mechanisms, such as changes in synapses, were not observed in vivo Overall, we demonstrated with our method rapid genetic manipulation and analysis of neuronal activities as well as precision transgene expression.

Development of a thermostable Cre/lox-based gene disruption system and in vivo manipulations of the megaplasmid pTT27 in Thermus thermophilus HB27.

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and ß-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.

Towards a Light-mediated Gene Therapy for the Eye using Caged Ethinylestradiol and the Inducible Cre/lox System.

Increasingly, retinal pathologies are being treated with virus-mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e., "caging." Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP-CreERT2 mice with a Cre-dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light-mediated gene therapy for the eyes.

Cardiac-Specific Cre Induces Age-Dependent Dilated Cardiomyopathy (DCM) in Mice.

The genetic modification of the mouse genome using the cre-lox system has been an invaluable tool in deciphering gene and protein function in a temporal and/or spatial manner. However, it has its pitfalls, as researchers have shown that the unregulated expression of cre recombinase can cause DNA damage, the consequences of which can be very detrimental to mouse health. Previously published literature on the most utilized cardiac-specific cre, αMHC-cre, mouse model exhibited a nonlethal hypertrophic cardiomyopathy (HCM) with aging. However, using the same αMHC-cre mice, we observed a cardiac pathology, resulting in complete lethality by 11 months of age. Echocardiography and histology revealed that the αMHC-cre mice were displaying symptoms of dilated cardiomyopathy (DCM) by seven months of age, which ultimately led to their demise in the absence of any HCM at any age. Molecular analysis showed that this phenotype was associated with the DNA damage response through the downregulation of activated p38 and increased expression of JNK, p53, and Bax, known inducers of myocyte death resulting in fibrosis. Our data urges strong caution when interpreting the phenotypic impact of gene responses using αMHC-cre mice, since a lethal DCM was induced by the cre driver in an age-dependent manner in this commonly utilized model system.

Coupling the recombineering to Cre-lox system enables simplified large-scale genome deletion in Lactobacillus casei.

BACKGROUND: Lactobacillus casei is widely used in the dairy and pharmaceutical industries and a promising candidate for use as cell factories. Recently, genome sequencing and functional genomics provide the possibility for reducing L. casei genome. However, it was still limited by the inefficient and laborious genome deletion methods. RESULTS: Here, we proposed a genome minimization strategy based on LCABL_13040-50-60 recombineering and Cre-lox site-specific recombination system in L. casei. The LCABL_13040-50-60 recombineering system was used to introduce two lox sites (lox66 and lox71) into 5' and 3' ends of the targeted region. Subsequently, the targeted region was excised by Cre recombinase. The robustness of the strategy was demonstrated by single-deletion of a nonessential ~ 39.3 kb or an important ~ 12.8 kb region and simultaneous deletion of two non-continuous genome regions (5.2 and 6.6 kb) with 100% efficiency. Furthermore, a cyclical application of this strategy generated a double-deletion mutant of which 1.68% of the chromosome was sequentially excised. Moreover, biological features (including growth rate, electroporation efficiency, cell morphology or heterologous protein productivity) of these mutants were characterized. CONCLUSIONS: To our knowledge, this strategy is the first instance of sequential deletion of large-scale genome regions in L. casei. We expected this efficient and inexpensive tool can help for rapid genome streamlining and generation restructured L. casei strains used as cell factories.

Cre-inducible site-specific recombination in zebrafish oligodendrocytes.

BACKGROUND: The conditional Cre/lox system has recently emerged as a valuable tool for studies on both embryonic and adult Zebrafish. Temporal control and site-specific recombination are achieved by using the ligand-inducible CreERT2 and administration of the drug tamoxifen (TAM) or its active metabolite, 4-Hydroxytamoxifen (4-OHT). RESULTS: Here we report the generation of a transgenic Zebrafish line, which expresses an mCherry-tagged variant of CreERT2 under the control of the myelin basic protein a (mbpa) promoter. Our analysis shows that larval and adult expression of the transgene recapitulates the endogenous mbpa expression pattern in oligodendrocytes. Furthermore, combination with a Cre-dependent EGFP reporter results in EGFP-expressing oligodendrocytes in the spinal cord, brain, and optic nerve in TAM- or 4-OHT-treated larvae and 4-month-old fish, but not in untreated controls. CONCLUSIONS: The transgenic Zebrafish line Tg(mbpa:mCherry-T2A-CreERT2 ) elicits CreERT2 expression specifically in myelinating glia cells. Cre-inducible targeted recombination of genes in oligodendrocytes will be useful to elucidate cellular and molecular mechanisms of myelination in vivo during development (myelination) and regeneration (remyelination) after injury to the central nervous system (CNS). It will also allow targeted expression and overexpression of genes of interest (transgenes) in oligodendrocytes at defined developmental and adult stages. Developmental Dynamics 246:41-49, 2017. © 2016 Wiley Periodicals, Inc.

Identification of clinical target areas in the brainstem of prion-infected mice.

AIMS: While prion infection ultimately involves the entire brain, it has long been thought that the abrupt clinical onset and rapid neurological decline in laboratory rodents relates to involvement of specific critical neuroanatomical target areas. The severity and type of clinical signs, together with the rapid progression, suggest the brainstem as a candidate location for such critical areas. In this study we aimed to correlate prion pathology with clinical phenotype in order to identify clinical target areas. METHOD: We conducted a comprehensive survey of brainstem pathology in mice infected with two distinct prion strains, which produce different patterns of pathology, in mice overexpressing prion protein (with accelerated clinical onset) and in mice in which neuronal expression was reduced by gene targeting (which greatly delays clinical onset). RESULTS: We identified specific brainstem areas that are affected by prion pathology during the progression of the disease. In the early phase of disease the locus coeruleus, the nucleus of the solitary tract, and the pre-Bötzinger complex were affected by prion protein deposition. This was followed by involvement of the motor and autonomic centres of the brainstem. CONCLUSIONS: Neurodegeneration in the locus coeruleus, the nucleus of the solitary tract and the pre-Bötzinger complex predominated and corresponded to the manifestation of the clinical phenotype. Because of their fundamental role in controlling autonomic function and the overlap with clinical signs in sporadic Creutzfeldt-Jakob disease, we suggest that these nuclei represent key clinical target areas in prion diseases.

Zona Glomerulosa-Derived Klotho Modulates Aldosterone Synthase Expression in Young Female Mice.

Klotho plays a critical role in the regulation of ion and fluid homeostasis. A previous study reported that haplo-insufficiency of Klotho in mice results in increased aldosterone synthase (CYP11B2) expression, elevated plasma aldosterone, and high blood pressure. This phenotype was presumed to be the result of diminished Klotho expression in zona glomerulosa (zG) cells of the adrenal cortex; however, systemic effects on adrenal aldosterone production could not be ruled out. To examine whether Klotho expressed in the zG is indeed a critical regulator of aldosterone synthesis, we generated a tamoxifen-inducible, zG-specific mouse model of Klotho deficiency by crossing Klotho-flox mice with Cyp11b2-CreERT mice (zG-Kl-KO). Tamoxifen-treated Cyp11b2-CreERT animals (zG-Cre) served as controls. Rosa26-mTmG reporter mice were used for Cre-dependent lineage-marking. Two weeks after tamoxifen induction, the specificity of the zG-Cre line was verified using immunofluorescence analysis to show that GFP expression was restricted to the zG. RNA in situ hybridization revealed a 65% downregulation of Klotho messenger RNA expression in the zG of zG-Kl-KO female mice at age 12 weeks compared to control mice. Despite this significant decrease, zG-Kl-KO mice exhibited no difference in plasma aldosterone levels. However, adrenal CYP11B2 expression and the CYP11B2 promotor regulatory transcription factors, NGFIB and Nurr1, were enhanced. Together with in vitro experiments, these results suggest that zG-derived Klotho modulates Cyp11b2 but does not evoke a systemic phenotype in young adult mice on a normal diet. Further studies are required to investigate the role of adrenal Klotho on aldosterone synthesis in aged animals.

Rodent models of tumours of the central nervous system.

Modelling of human diseases is an essential component of biomedical research, to understand their pathogenesis and ultimately, develop therapeutic approaches. Here, we will describe models of tumours of the central nervous system, with focus on intrinsic CNS tumours. Model systems for brain tumours were established as early as the 1920s, using chemical carcinogenesis, and a systematic analysis of different carcinogens, with a more refined histological analysis followed in the 1950s and 1960s. Alternative approaches at the time used retroviral carcinogenesis, allowing a more topical, organ-centred delivery. Most of the neoplasms arising from this approach were high-grade gliomas. Whilst these experimental approaches did not directly demonstrate a cell of origin, the localisation and growth pattern of the tumours already pointed to an origin in the neurogenic zones of the brain. In the 1980s, expression of oncogenes in transgenic models allowed a more targeted approach by expressing the transgene under tissue-specific promoters, whilst the constitutive inactivation of tumour suppressor genes ('knock out')-often resulted in embryonic lethality. This limitation was elegantly solved by engineering the Cre-lox system, allowing for a promoter-specific, and often also time-controlled gene inactivation. More recently, the use of the CRISPR Cas9 technology has significantly increased experimental flexibility of gene expression or gene inactivation and thus added increased value of rodent models for the study of pathogenesis and establishing preclinical models.

Lineage Tracing Methods to Study Mammary Epithelial Hierarchies In Vivo.

Lineage tracing is now considered the gold standard approach to study cellular hierarchies and cell fate in vivo (McKenna and Gagnon, Development 146:dev169730, 2019; Kretzschmar and Watt, Cell 148:33-45, 2012). This type of clonal analysis consists of genetically labeling defined cells and following their destiny and progeny in vivo and in situ.Here we will describe different existing in vivo systems to clonally trace targeted cells and will discuss their respective advantages and inconveniences; we will then provide stepwise instructions for setting up and evaluate lineage tracing experiments, listing the most common downstream analyses and read-out assays.

Generating Rab6 Conditional Knockout Mice.

RAB6 GTPase is the most abundant Golgi-associated RAB protein and regulates several transport steps at the level of this organelle. Homozygous Rab6a knockout (k/o) is embryonic lethal in mouse. To study RAB6 function in cell lineages and tissues, we thus generated various conditional Rab6a knockout (k/o) mice using the Cre/lox system.

Cartilage-Specific Cre Recombinase Transgenes/Alleles in the Mouse.

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.

Eliminating the VGlut2-Dependent Glutamatergic Transmission of Parvalbumin-Expressing Neurons Leads to Deficits in Locomotion and Vocalization, Decreased Pain Sensitivity, and Increased Dominance.

The calcium-binding protein parvalbumin (PV) is a recognized marker of short-axon GABA-ergic neurons in the cortex and the hippocampus. However in addition, PV is expressed by excitatory, glutamatergic neurons in various areas of the brain and spinal cord. Depending on the location of these neurons, loading of their synaptic vesicles with glutamate is mediated by either of three vesicular glutamate transporters (VGlut): VGlut1, VGlut2, or VGlut3. Driven by our interest in one of these glutamatergic/PV-expressing cell clusters-the lateral hypothalamic parvafox nucleus-we investigated the functions of this population of neurons by the selective deletion of VGlut2 expression in PV-expressing cells according to the Cre/Lox-approach. PV-Cre;VGlut2-Lox mutant mice are phenotypically characterized by deficits in locomotion and vocalization, by a decreased thermal nociception, and by an increased social dominance. We conducted a search of the Allen Brain Atlas for regions that might co-express the genes encoding PV and VGlut2, and that might thus contribute to the manifestation of the observed phenotypes. Our survey revealed several structures that could contribute to the deficits in locomotion and vocalization, such as the red, the subthalamic and the deep cerebellar nuclei. It also disclosed that a shift in the balance of afferental glutamatergic neurotransmission to the periaqueductal gray matter might be accountable for the decrease in sensitivity to pain and for the increase in social dominance. As a whole, this study broadens the state of knowledge about PV-expressing excitatory neurons.

A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines.

Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.

Site-specific transfer of chromosomal segments and genes in wheat engineered chromosomes.

Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited. Here, telomere-mediated truncation was successfully performed in common wheat (Triticum aestivum) to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.

A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker.

BACKGROUND: Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis. RESULTS: In this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d-alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46 U/ml at 72 h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5 l fermentor with a fed-batch fermentation. CONCLUSION: The alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis.

Site-specific recombinatorics: in situ cellular barcoding with the Cre Lox system.

BACKGROUND: Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues. RESULTS: With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice. CONCLUSIONS: Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.

Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system.

Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based Targeted Transgenesis (PITT), a method that uses a recombinase-mediated cassette exchange (RMCE) to enable insertion of sequences at a predetermined genomic locus, such as ROSA26. The PITT technique uses fertilized eggs (instead of ES cells) collected from 'seed mice' that contain the RMCE landing pad. The PITT method can rapidly generate reliable targeted transgenic mice; it requires a seed mouse, which in our previous study was generated using ES cell targeting approaches. Here, we demonstrate that seed mice containing the RMCE landing pad can be developed rapidly by using the CRISPR/Cas9 system. One of the CRISPR targets tested in this study enabled the insertion of sequences precisely at the original ROSA26 provirus integration site. We anticipate that using a similar approach, PITT landing pad sequences can be rapidly and precisely inserted at other genomic loci to develop an array of PITT tools. This two-step strategy combines the best features of the two newer technologies-rapid creation of PITT landing pads using the CRISPR/Cas9 system and efficient and precise insertion of larger cassettes at the landing pads using PITT. This study also revealed that anomalous and mosaic sequence insertions can occur with the CRISPR/Cas9 system.

Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis.

The lack of knowledge about pathogenicity mechanisms of Streptococcus (S.) suis is, at least partially, attributed to limited methods for its genetic manipulation. Here, we established a Cre-lox based recombination system for markerless gene deletions in S. suis serotype 2 with high selective pressure and without undesired side effects.