Comparative analysis of two independent Myh6-Cre transgenic mouse lines.

We have previously shown that the Myh6 promoter drives Cre expression in a subset of male germ line cells in three independent Myh6-Cre mouse lines, including two transgenic lines and one knock-in allele. In this study, we further compared the tissue-specificity of the two Myh6-Cre transgenic mouse lines, MDS Myh6-Cre and AUTR Myh6-Cre, through examining the expression of tdTomato (tdTom) red fluorescence protein in multiple internal organs, including the heart, brain, liver, lung, pancreas and brown adipose tissue. Our results show that MDS Myh6-Cre mainly activates tdTom reporter in the heart, whereas AUTR Myh6-Cre activates tdTom expression significantly in the heart, and in the cells of liver, pancreas and brain. In the heart, similar to MDS Myh6-Cre, AUTR Myh6-Cre activates tdTom in most cardiomyocytes. In the other organs, AUTR Myh6-Cre not only mosaically activates tdTom in some parenchymal cells, such as hepatocytes in the liver and neurons in the brain, but also turns on tdTom in some interstitial cells of unknown identity.

Platform establishment of the Cre-loxP recombination system for genetic manipulation of the Lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Genetic manipulation of the LSDV is essential for the elucidation of the pathogenic mechanism and biological function of the LSDV-encoded protein. In this study, we established a platform for the Cre-loxP recombination system under a modified early-late H5 promoter of the VACV for quick construction of the recombinant LSDV virus. The recombinant virus, LSDV-EGFP-ΔTK, was purified and obtained using serial limited dilution and picking the single cells methods. Using the lentiviral package system, a Cre recombinase enzyme stable expression MDBK cell line was established to supply the Cre recombinase for the reporter gene excision. A genetically stable, safe TK gene-deleted LSDV (LSDV-ΔTK) was constructed using homologous recombination and the Cre-loxP system. It was purified using limited dilution in the MDBK-Cre cell line. Establishing the Cre-loxP recombination system will enable sequential deletion of the interested genes from the LSDV genome and genetic manipulation of the LSDV genome, providing technical support and a platform for developing the attenuated LSDV vaccine.

An Hsp70 promoter-based mouse for heat shock-induced gene modulation.

Physical therapy is extensively employed in clinical settings. Nevertheless, the absence of suitable animal models has resulted in an incomplete understanding of the in vivo mechanisms and cellular distribution that respond to physical stimuli. The objective of this research was to create a mouse model capable of indicating the cells affected by physical stimuli. In this study, we successfully established a mouse line based on the heat shock protein 70 (Hsp70) promoter, wherein the expression of CreERT2 can be induced by physical stimuli. Following stimulation of the mouse tail, ear, or cultured calvarias with heat shock (generated by heating, ultrasound, or laser), a distinct Cre-mediated excision was observed in cells stimulated by these physical factors with minimal occurrence of leaky reporter expression. The application of heat shock to Hsp70-CreERT2; FGFR2-P253R double transgenic mice or Hsp70-CreERT2 mice infected with AAV-BMP4 at calvarias induced the activation of Cre-dependent mutant FGFR2-P253R or BMP4 respectively, thereby facilitating the premature closure of cranial sutures or the repair of calvarial defects. This novel mouse line holds significant potential for investigating the underlying mechanisms of physical therapy, tissue repair and regeneration, lineage tracing, and targeted modulation of gene expression of cells in local tissue stimulated by physical factor at the interested time points. KEY MESSAGES: In the study, an Hsp70-CreERT2 transgenic mouse was generated for heat shock-induced gene modulation. Heat shock, ultrasound, and laser stimulation effectively activated Cre expression in Hsp70-CreERT2; reporter mice, which leads to deletion of floxed DNA sequence in the tail, ear, and cultured calvaria tissues of mice. Local laser stimuli on cultured calvarias effectively induce Fgfr2-P253R expression in Hsp70-mTmG-Fgfr2-P253R mice and result in accelerated premature closure of cranial suture. Heat shock activated AAV9-FLEX-BMP4 expression and subsequently promoted the repair of calvarial defect of Hsp70-CreERT2; Rosa26-mTmG mice.

Tissue-specific Cre driver mice to study vascular diseases.

Vascular diseases, including atherosclerosis and abdominal aneurysms, are the primary cause of mortality and morbidity among the elderly worldwide. The life quality of patients is significantly compromised due to inadequate therapeutic approaches and limited drug targets. To expand our comprehension of vascular diseases, gene knockout (KO) mice, especially conditional knockout (cKO) mice, are widely used for investigating gene function and mechanisms of action. The Cre-loxP system is the most common method for generating cKO mice. Numerous Cre driver mice have been established to study the main cell types that compose blood vessels, including endothelial cells, smooth muscle cells, and fibroblasts. Here, we first discuss the characteristics of each layer of the arterial wall. Next, we provide an overview of the representative Cre driver mice utilized for each of the major cell types in the vessel wall and their most recent applications in vascular biology. We then go over Cre toxicity and discuss the practical methods for minimizing Cre interference in experimental outcomes. Finally, we look into the future of tissue-specific Cre drivers by introducing the revolutionary single-cell RNA sequencing and dual recombinase system.

Recent advances in in situ Notch signaling measurement.

Notch signaling is necessary for the development of many organ systems, including the nervous system, biliary system, and visual and auditory sensory systems. This signaling pathway is composed of DSL ligands and Notch receptors. Upon the interaction of those components between neighboring cells, the intracellular domain of the Notch receptor is cleaved from the cell membrane to act as a transcription factor. To date, many mechanistic insights, including lateral inhibition and lateral induction, have been proposed from observation of patterning morphogenesis and expression profiles of Notch signaling-associated molecules. The lack of a direct measurement method for Notch signaling, however, has impeded the examination of those mechanistic insights. In this mini-review, recent advances in the direct measurement of Notch signaling are introduced with a focus on the application of genetic modification of Notch receptors with the components of the Cre/loxP system and Gal4/UAS system. The combination of such conventional genetic techniques is opening a new era in Notch signaling biology by direct visualization of Notch "signaling" in addition to Notch signaling-associated molecules.

Analysis of Notch1 signaling in mammalian sperm development.

OBJECTIVE: A mammalian Delta-Notch signaling component, Notch1, has been suggested for its expression during the normal sperm development although its conditional deletion caused no apparent abnormalities. Since we established our original transgenic mouse system that enabled labeling of past and ongoing Notch1 signaling at a cellular level, we tried to validate that observation in vivo. Our transgenic mouse system used Cre/loxP system to induce tandem dsRed expression upon Notch1 signaling. RESULTS: To our surprise, we were unable to observe tandem dsRed expression in the seminiferous tubules where the sperms developed. In addition, tandem dsRed expression was lacking in the somatic cells of the next generation in our transgenic mouse system, suggesting that sperms received no Notch1 signaling during their development. To validate this result, we conducted re-analysis of four single-cell RNA-seq datasets from mouse and human testes and showed that Notch1 expression was little in the sperm cell lineage. Collectively, our results posed a question into the involvement of Notch1 in the normal sperm development although this observation may help the interpretation of the previous result that Notch1 conditional deletion caused no apparent abnormalities in murine spermatogenesis.

A review of tamoxifen administration regimen optimization for Cre/loxp system in mouse bone study.

Gene knockout is a technique routinely used in basic experimental research, particularly in mouse skeletal and developmental studies. Tamoxifen-induced Cre/loxp system is known for its temporal and spatial precision and commonly utilized by researchers. However, tamoxifen has been shown its side effects on affecting the phenotype of mouse bone directly. This review aimed to optimize tamoxifen administration regimens including its dosage and duration, to identify an optimal induction strategy that minimizes potential side effects while maintaining recombination efficacy. This study will help researchers in designing gene knockout experiments in bone when using tamoxifen.

Notch1 signaling is limited in healthy mature kidneys in vivo.

OBJECTIVE: A Delta-Notch signaling component, Notch1, is involved in the normal development and multiple disorders of the kidney. Although the increase in Notch1 signaling is crucial to these pathogeneses, the basal signaling level in 'healthy' mature kidneys is still unclear. To address this question, we used an artificial Notch1 receptor fused with Gal4/UAS components in addition to the Cre/loxP system and fluorescent proteins in mice. This transgenic reporter mouse system enabled labeling of past and ongoing Notch1 signaling with tdsRed or Cre recombinase, respectively. RESULTS: We confirmed that our transgenic reporter mouse system mimicked the previously reported Notch1 signaling pattern. Using this successful system, we infrequently observed cells with ongoing Notch1 signaling only in Bowman's capsule and tubules. We consider that Notch1 activation in several lines of disease model mice was pathologically significant itself.

Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer.

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine ß-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

A Cre-LoxP-based approach for combinatorial chromosome rearrangements in human HAP1 cells.

Alterations of human karyotype caused by chromosomal rearrangements are often associated with considerable phenotypic effects. Studying molecular mechanisms underlying these effects requires an efficient and scalable experimental model. Here, we propose a Cre-LoxP-based approach for the generation of combinatorial diversity of chromosomal rearrangements. We demonstrate that using the developed system, both intra- and inter-chromosomal rearrangements can be induced in the human haploid HAP1 cells, although the latter is significantly less effective. The obtained genetically modified HAP1 cell line can be used to dissect genomic effects associated with intra-chromosomal structural variations.

Analysis of the Function of CCN2 in Tubular Epithelium Cells with a Focus on Renal Fibrogenesis.

Renal interstitial fibrosis is the final common pathway in the process of all kidney diseases, and it results in chronic kidney disease. CCN2 is an important factor in the pathogenesis of renal interstitial fibrosis, and analysis of its function can lead to treatments for chronic kidney disease. Since CCN2 knockout mice are developmentally lethal, generation of conditional knockout mice is essential for in vivo analysis. Since CCN2 is expressed in a variety of cells in the kidney, including podocytes, mesangial cells, pericytes, and tubular epithelial cells, it is necessary to perform cell-specific verification of the cells that play a central role in fibrosis. However, cell-specific validation using the Cre/loxP system in vivo has only been performed in mesangial cells. In our research program, we are focusing on the role of CCN2 in tubular epithelial cells in renal fibrogenesis. In this report, we introduce the creation of a tubular epithelial cell-specific knockout model and method of its analysis.

Generation of Conditional KO Mice of CCN2 and Its Function in the Kidney.

CCN2 has been shown to be closely involved in the progression of renal fibrosis, indicating the potential of CCN2 inhibition as a therapeutic target. Although the examination of the renal disease phenotypes of adult CCN2 knockout mice has yielded valuable scientific insights, perinatal death has limited studies of CCN2 in vivo. Conditional knockout technology has become widely used to delete genes in the target cell populations or time points using cell-specific Cre recombinase-expressing mice. Therefore, several lines of CCN2-floxed mice have been developed to assess the functional role of CCN2 in adult mice.CCN2 levels are elevated in renal fibrosis and proliferative glomerulonephritis, making them suitable disease models for assessing the effects of CCN2 deletion on the kidney. Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis and transforming growth factor-ß. CCN2 is increased in fibrosis and modulates a number of downstream signaling pathways involved in the fibrogenic properties of TGF-ß. Unilateral ureteral obstruction is one of the most widely used models of renal tubulointerstitial fibrosis. In addition, anti-glomerular basement membrane antibody glomerulonephritis has become the most widely used model for evaluating the effect of increased renal CCN2 expression. Herein, we describe the construction of CCN2-floxed mice and inducible systemic CCN2 conditional knockout mice and methods for the operation of unilateral ureteral obstruction and the induction of anti-glomerular basement membrane antibody glomerulonephritis.

Cre-recombinase systems for induction of neuron-specific knockout models: a guide for biomedical researchers.

Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology. Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location, respectively. Using these technologies in mouse embryos led to the generation of mouse knockout models and many scientific discoveries. The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as clustered regularly interspaced short palindromic repeats for targetted gene deletion. However, several limitations including unwanted off-target gene deletion have hindered their widespread use in the field. Cre-recombinase technology has provided additional capacity for cell-specific gene deletion. In this review, we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes. This article has been constructed to provide some background information for the new trainees on the mechanism and to provide necessary information for the design, and application of the Cre-recombinase system through reviewing the most frequent promoters that are currently available for genetic manipulation of neurons. We additionally will provide a summary of the latest technological developments that can be used for targeting neurons. This may also serve as a general guide for the selection of appropriate models for biomedical research.

Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche.

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Red blood cell eNOS is cardioprotective in acute myocardial infarction.

Red blood cells (RBCs) were shown to transport and release nitric oxide (NO) bioactivity and carry an endothelial NO synthase (eNOS). However, the pathophysiological significance of RBC eNOS for cardioprotection in vivo is unknown. Here we aimed to analyze the role of RBC eNOS in the regulation of coronary blood flow, cardiac performance, and acute myocardial infarction (AMI) in vivo. To specifically distinguish the role of RBC eNOS from the endothelial cell (EC) eNOS, we generated RBC- and EC-specific knock-out (KO) and knock-in (KI) mice by Cre-induced inactivation or reactivation of eNOS. We found that RBC eNOS KO mice had fully preserved coronary dilatory responses and LV function. Instead, EC eNOS KO mice had a decreased coronary flow response in isolated perfused hearts and an increased LV developed pressure in response to elevated arterial pressure, while stroke volume was preserved. Interestingly, RBC eNOS KO showed a significantly increased infarct size and aggravated LV dysfunction with decreased stroke volume and cardiac output. This is consistent with reduced NO bioavailability and oxygen delivery capacity in RBC eNOS KOs. Crucially, RBC eNOS KI mice had decreased infarct size and preserved LV function after AMI. In contrast, EC eNOS KO and EC eNOS KI had no differences in infarct size or LV dysfunction after AMI, as compared to the controls. These data demonstrate that EC eNOS controls coronary vasodilator function, but does not directly affect infarct size, while RBC eNOS limits infarct size in AMI. Therefore, RBC eNOS signaling may represent a novel target for interventions in ischemia/reperfusion after myocardial infarction.

Downregulation of eNOS and preserved endothelial function in endothelial-specific arginase 1-deficient mice.

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.

Elimination of Reprogramming Transgenes Facilitates the Differentiation of Induced Pluripotent Stem Cells into Hepatocyte-like Cells and Hepatic Organoids.

Hepatocytes and hepatic organoids (HOs) derived from human induced pluripotent stem cells (hiPSCs) are promising cell-based therapies for liver diseases. The removal of reprogramming transgenes can affect hiPSC differentiation potential into the three germ layers but not into hepatocytes and hepatic organoids in the late developmental stage. Herein, we generated hiPSCs from normal human fibroblasts using an excisable polycistronic lentiviral vector based on the Cre recombinase-mediated removal of the loxP-flanked reprogramming cassette. Comparing the properties of transgene-carrying and transgene-free hiPSCs with the same genetic background, the pluripotent states of all hiPSCs were quite similar, as indicated by the expression of pluripotent markers, embryonic body formation, and tri-lineage differentiation in vitro. However, after in vitro differentiation into hepatocytes, transgene-free hiPSCs were superior to the transgene-residual hiPSCs. Interestingly, the generation and hepatic differentiation of human hepatic organoids (hHOs) were significantly enhanced by transgene elimination from hiPSCs, as observed by the upregulated fetal liver (CK19, SOX9, and ITGA6) and functional hepatocyte (albumin, ASGR1, HNF4α, CYP1A2, CYP3A4, and AAT) markers upon culture in differentiation media. Thus, the elimination of reprogramming transgenes facilitates hiPSC differentiation into hepatocyte-like cells and hepatic organoids with properties of liver progenitor cells. Our findings thus provide significant insights into the characteristics of iPSC-derived hepatic organoids.

Identification of the Transgene Integration Site and Host Genome Changes in MRP8-Cre/ires-EGFP Transgenic Mice by Targeted Locus Amplification.

The MRP8-Cre-ires/EGFP transgenic mouse (Mrp8creTg, on C57BL/6J genetic background) is popular in immunological and hematological research for specifically expressing Cre recombinase and an EGFP reporter in neutrophils. It is often crossed with other transgenic lines carrying loxP-flanked genes to achieve restricted gene knockout in neutrophils. However, due to the way in which the line was created, basic knowledge about the MRP8-Cre-ires/EGFP transgene in the host genome, such as its integration site(s) and flanking sequences, remains largely unknown, hampering robust experimental design and data interpretation. Here we used a recently developed technique, targeted locus amplification (TLA) sequencing, to fill these knowledge gaps. We found that the MRP8-Cre-ires/EGFP transgene was integrated into chromosome 5 (5qG2) of the host mouse genome. This integration led to a 44 kb deletion of the host genomic sequence, resulting in complete deletion of Serpine1 and partial deletion of Ap1s1. Having determined the flanking sequences of the transgene, we designed a new genotyping protocol that can distinguish homozygous, heterozygous, and wildtype Mrp8creTg mice. To our surprise, crossing heterozygous mice produced no homozygous Mrp8creTg mice, most likely due to prenatal lethality resulting from disrupted Ap1s1 gene expression.