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.

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.

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-β. CCN2 is increased in fibrosis and modulates a number of downstream signaling pathways involved in the fibrogenic properties of TGF-β. 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.

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.

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.

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.

Mutations in the CRB1 (Crumbs homolog 1) cause rare retinal diseases like retinitis pigmentosa type 12 (RP12) and Leber congenital amaurosis type 8 (LCA8). RP12 results in progressively worsening peripheral vision, whereas LCA8 causes severe visual impairment at birth or in early life. While several mouse models have been proposed for RP12, few replicate the full spectrum of human LCA8 pathology, such as disorganized retinal layering, abnormal retinal thickening, pigmentary defects, hyperreflective lesions, and severely attenuated electroretinogram responses at birth. Six models have been proposed utilizing the Cre-loxP system to delete candidate genes in specific retinal cell types and developmental stages. The model ablating Crb1 and its homolog Crb2 (using mRx-Cre) from the beginning of the eye development is the most complete as it shows blindness during the eye-opening stage, pigmentary defects in the RPE, ganglion cell layer heterotopia, disruption of retinal lamination, and acellular patches. LCA8 represents a unique type of retinal dystrophy among LCA subtypes, driven by dysfunctional retinal progenitor cells during eye development. In contrast, other LCA types and RP12 are caused by photoreceptor defects. Therefore, the most accurate LCA8-like mouse model must target both alleles of the Crb1 and Crb2 genes in the optic vesicle or earlier.

Mammalian kidneys consist of more than 30 different types of cells. A challenging task is to identify and characterize the stem/progenitor subpopulations that establish the lineage relationships among these cellular elements during nephrogenesis in the embryonic and neonate kidneys and during tissue homeostasis and/or injury repair in the mature kidney. Moreover, the potential clinical utility of stem/progenitor cells holds promise for the development of new regenerative medicine approaches for the treatment of renal diseases. Stem cells are defined by unlimited self-renewal capacity and pluripotentiality. Progenitor cells have pluripotentiality but no or limited self-renewal potential. Cre-LoxP-based in vivo genetic lineage tracing is a powerful tool to identify stem/progenitor cells in their native environment. Hypothetically, this technique enables investigators to accurately track the progeny of a single cell or a group of cells. The Cre/LoxP system has been widely used to uncover the function of genes in various mammalian tissues and to identify stem/progenitor cells through in vivo lineage tracing analyses. In this review, we summarize the recent advances in the development and characterization of various Cre drivers and their use in identifying potential renal stem/progenitor cells in both developing and mature mouse kidneys.

The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.

In mice, the conditional knockout strategy using the Cre-loxP system is useful for various types of research. The Cre mouse line with progesterone receptor promoter (PgrCre ) has been widely used to produce specific uterine gene-deficient mice, but in the Cre line, endogenous Pgr gene is replaced by Cre recombinase gene, which makes the breeding of homozygous mice (PgrCre/Cre ) difficult because they are infertile. Yang et al. (2013, https://10.1016/j.cell.2013.04.017) reported the generation of another PgriresCre mouse line that still has endogenous Pgr gene, and they inserted Cre recombinase downstream of the Pgr gene via an internal ribosome entry site (IRES). It is possible that this new PgriresCre line would be useful for uterine research as the mice can be bred as homozygotes (PgriresCre/iresCre ). Herein, we confirmed the PgriresCre mice effectively directed recombination in the female reproductive tract and was capable of genetic alteration in the endometrium that enables the studies of its uterine function. Our findings demonstrate that the new PgriresCre mouse line is also useful for the generation of uterine-specific knockout mice. The findings using PgriresCre mouse will contribute to the understanding of reproductive systems and diseases in humans and domestic animals.