Cre Recombinase Protein Research Articles

Abstract B056: Selective epithelial activation of KRAS G12D mutation drives ductal pancreatic neoplasia in pigs

Abstract An innovative approach in pancreatic cancer (PC) research is to utilize large animal models which (i) would be useful for development of technologies that require a human-sized platform and (ii) would produce data that could have greater clinical relevance and accuracy compared to data produced by murine models. To this end, our group has been developing porcine PC models and has reliably induced PC in the KRAS G12D/p53 R167H Oncopig model. The objective of the present study was to generate a slower-growing, early-stage PC model with intraductal injection of adenoviral vector with a Keratin 8 promoter driving the expression of the Cre-Recombinase protein (Ad5mK8-nlsCre) in oncopigs which, we hypothesized, would produce pancreatic neoplasia restricted to the epithelium. Results We first confirmed with scRNA-seq and immunohistochemistry that Keratin-8 expression in the porcine pancreas was restricted to ductal and acinar cells. Using an ex vivo infection assay with the Ad5mK8-nlsCre vector in wild-type porcine pancreas, we then demonstrated that expression of Cre-recombinase was confined to epithelial cells. We then performed a dose-response study of ductal injection (laparotomy) of Ad5mK8-nlsCre (10(7)-10(10) pfu) in Oncopigs. At the planned 2-month endpoint, all Ad5mK8-nlsCre concentrations produced microscopic evidence of ductal hyperplasia, metaplasia, and nuclear atypia, with KRAS-G12D expression by immunohistochemistry. At the higher Ad5mK8-nlsCre concentration, there was increased ductal mucin production. Using an epithelial-specific AdCre vector, we produced microscopic epithelial neoplasia within the pancreas of the Oncopig. Conclusion While further follow-up and characterization is needed, this induction technique may yield a porcine model of early-stage PC, which could be useful to (i) study early tumor biology and progression and (ii) develop diagnostic and interventional technologies for early tumor management. Citation Format: Carlos P. Jara, Al-Murtadha Al-Gahmi, Binh Le, Mark A. Carlson. Selective epithelial activation of KRAS G12D mutation drives ductal pancreatic neoplasia in pigs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B056.

FRI533 A Novel Cre-loxP Based Mouse Model For Graves' Disease

Abstract Disclosure: D. Kim: None. Y. Bao: None. M. Kang: None. Y. Cho: None. Y. Kim: None. Y. Hwang: None. C. Ku: None. E. Lee: None. Objectives: Over years, various approaches have been used to model human Graves’ disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunizations with the extracellular A subunit of the human thyrotropin receptor (hTSHRa). However, these models require a lot of effort for multiple dosing over several months and the results are very heterogeneous, limiting their use as a platform for the development of new therapeutics. Thus, we developed a novel Cre-loxP based mouse model for Graves' disease. Methods: To overcome the limitation of existing animal models, we made ESC-derived KI mouse expressing hTSHRa that will act as an antigen in a conditional manner by inserting the CAG-loxP-STOP-loxP-hTSHRa cassette into the mouse Rosa26 locus. TAT-Cre, a recombinant cell-permeant fusion cre-recombinase protein, was used for site-specific recombination to avoid leaky expression which may induce immune tolerance. Various concentrations of TAT-Cre were tested to avoid neutralization and produce adequate hyperthyroidism. Serum TSHR Ab and T4 levels were measured. Immunohistochemical examinations of the thyroid gland and orbits were performed. Results: Treatment with TAT-Cre within the range of 250-750U produced the most efficient TSHR Ab and significantly increased T4. Four weeks after TAT-Cre 500U administration, TSHR Ab levels were already significantly produced in all mice and T4 levels started to increase. At 8 and 12 weeks, TSHR Ab levels were produced at a very high levels in all mice and T4 levels were increased to a very high levels in over 80% of mice. After 3 months, MRI images did not show any significant protrusion. However, there was an enlargement in the volume of the orbital muscles. We also found thyroid enlargement and hypertrophy, orbital macrophage infiltration and fibrosis in Immunohistochemical examinations. Conclusions: A new mouse model for Graves' disease was successfully established by using the Cre-loxP system. After being optimized for orbitopathy induction, this model can be used as a platform for the development of novel therapeutics. Presentation: Friday, June 16, 2023

In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

Targeted Knockdown of Genes in the Choroid Plexus.

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

A Method for Electroporation of Cre Recombinase Protein into Intact Nicotiana tabacum Cells.

The Cre/lox recombination system has become a powerful technology for gene function analysis in a broad spectrum of cell types and organisms. In our previous report, Cre protein had been successfully delivered into intact Arabidopsis thaliana cells using electroporation. To expand the feasibility of the method of protein electroporation to other plant cells, here we attempt the protein electroporation into tobacco-derived BY-2 cells, one of the most frequently used plant cell lines for industrial production. In this study, we successfully deliver Cre protein into BY-2 cells with intact cell walls by electroporation with low toxicity. Targeted loxP sequences in the BY-2 genome are recombined significantly. These results provide useful information for genome engineering in diverse plant cells possessing various types of cell walls.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

Brd4 Inactivation Increases Adenoviral Delivery of BMP2 for Paracrine Stimulation of Osteogenic Differentiation as a Gene Therapeutic Concept to Enhance Bone Healing.

ABSTRACTBromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow–derived mesenchymal stem cells via a conditional Brd4fl/fl allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector‐based expression of BMP2 mRNA and protein levels are enhanced upon Brd4 depletion in cells transduced with an adenoviral vector that expresses BMP2 (Ad‐BMP2). Importantly, Brd4 depletion in MC3T3‐E1 and human adipose‐derived mesenchymal stem cells (AMSCs) transduced with Ad‐BMP2 enhances osteogenic differentiation of naïve MC3T3‐E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells, which can be leveraged as a therapeutic strategy to improve viral vector‐based gene therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

SPARC: Development of a Transgenic Pig Expressing mCherry and CRE Recombinase Fusion Proteins Under the Control of the Murine Choline Acetyltransferase Promoter

Pigs can serve as useful models for advancing the biomedical field due to anatomy and physiology that more closely resembles human anatomy and physiology compared to traditional rodent models. However, there is a paucity of porcine CRE-driver lines available, limiting their utility for refined neural mapping and neural modulation studies. Here we describe the development of pigs with mCherry and CRE recombinase proteins expressed in cholinergic neurons. A 6,419 bp DNA fragment containing the Mus musculus choline acetyltransferase (ChAT) promoter was used to drive production of a mCherry–P2A–CRE recombinase fusion protein (mmChAT–mCherry–P2A–CRE). Linearized mmChAT–mCherry–P2A–CRE plasmid DNA was introduced into male porcine fetal fibroblasts by electroporation. Positive colonies were selected with 400µg/mL G-418 and screened for transgene insertion by PCR. Two colonies showing targeted insertion were used for somatic cell nuclear transfer and the resulting blastocysts were transferred to surrogate gilts. Two early pregnancies were established, and one of them went to term. Six Duroc-Landrace male piglets were born by natural delivery. PCR analysis of genomic DNA showed that one piglet carried the transgene whereas the others were determined to be wild-type. The transgenic piglet was phenotypically normal with no obvious difference in appearance compared to the wild-type littermates. The piglet was euthanized at 7-weeks of age for further characterization. Major tissues known to have cholinergic cell bodies and cholinergic innervation (hippocampus, cerebellum, spinal cord, basal ganglion, dorsal root ganglion, nodose ganglion, vagus nerve, trachea, bronchus, heart, bladder, duodenum and pancreas) were evaluated regarding mCherry and CRE recombinase mRNA presence. End-point PCR revealed CRE recombinase mRNA in basal ganglion, nodose ganglion, dorsal root ganglion and heart. mCherry mRNA was found in bronchus homogenates. Studies are ongoing to confirm integration sites into the genome, and mCherry and CRE recombinase expression at the protein level in cholinergic neurons. If successful, this model will allow for simultaneous dissection of cholinergic circuit connectivity and control of neural activity through CRE-mediated recombination.

Endosomal escape enhancing compounds facilitate functional delivery of extracellular vesicle cargo.

Aim: Extracellular vesicles (EVs) are desirable delivery vehicles for therapeutic cargoes. We aimed to load EVs with Cre recombinase protein and determine whether functional delivery to cells could be improved by using endosomal escape enhancing compounds. Materials & methods: Overexpressed CreFRB protein was actively loaded into EVs by rapalog-induced dimerization to CD81FKBP, or passively loaded by overexpression in the absence of rapalog. Functional delivery of CreFRB was analysed using a HEK293 Cre reporter cell line in the absence and presence of endosomal escape enhancing compounds. Results:The EVs loaded with CreFRB by both active and passive mechanisms were able to deliver functional CreFRB to recipient cells only in the presence of endosomal escape enhancing compounds chloroquine and UNC10217938A. Conclusion:The use of endosomal escape enhancing compounds in conjunction with EVs loaded with therapeutic cargoes may improve efficacy of future EV based therapeutics.

Abstract 1765: In vivo modeling of RIT1 function in development and lung cancer

Abstract Ras-like in most tissues 1 (RIT1) encodes a ubiquitously expressed RAS-family small GTPase. Recently, somatic RIT1 mutations were identified in lung adenocarcinoma and myeloid malignancies, and germline RIT1 mutations were found in. RIT1 is also implicated hematopoietic malignancies and in Noonan syndrome, a “Rasopathy” affecting craniofacial development and associated with congenital heart disease. Of a variety of RIT1 mutations discovered, the most recurrent mutation observed was M90I. In this study, murine models were used to determine whether expression of RIT1M90I mutation is sufficient for tumor initiation or developmental phenotypes observed in Noonan patients. Mice were genetically engineered at the Rosa26 locus to harbor RIT1M90I under the control of a lox-Cre system (LSL-RIT1M90I). The Cre recombinase protein was introduced via intratracheal delivery of adenovirus to the lungs. At one-year post treatment, 7 of the 10 LSL-RIT1M90I mice examined exhibited the expected genetic recombination. Tumors were observed in 3 of these mice, one of which was wild-type for p53. No visible tumors were seen in mice lacking the RIT1M90I gene or in cases where recombination was not observed, regardless of p53 status. Mouse embryonic fibroblasts (MEFs) were infected by adenovirus to recapitulate the in vivo studies and evaluate transformation. In vitro experiments conducted in RIT1M90I MEFs demonstrated faithful recombination of the transgene upon treatment with Cre. Protein analysis of these same cells reflected higher levels of RIT1 protein in transgenic cells treated with Cre, while in all other conditions, RIT1 was undetectable. Preliminary data from an immortalization assay suggested enhanced proliferation of MEFs expressing the RIT1M90I gene compared to wild-type counterparts. Cre recombinase was expressed body-wide in tamoxifen (4OHT) inducible mice (UBC-Cre-ERT2) LSL-RIT1M90I animals crossed to the inducible UBC-Cre-ERT2 system exhibited faithful recombination of the transgene upon treatment with 4OHT. To determine the consequences of constitutive expression of RIT1M90I, RIT1M90I sires were crossed to dams harboring constitutively active Cre recombinase or to a constitutively active Cre SRY-box promoter (Sox2-Cre). Of the 100 pups observed, none contained both RIT1M90I and Cre recombinase (p < 0.0001), indicating embryonic lethality induced by RIT1 activation. These data demonstrate the powerful effect of RIT1 activation on murine embryonic development and will enable future efforts on the characterization of the mechanism of RIT1-induced Noonan Syndrome and lung cancer. Citation Format: Maria McSharry. In vivo modeling of RIT1 function in development and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1765.

Next Generation Transgenic Rat Model Production.

The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.

New Plasmids for Fusarium Transformation Allowing Positive-Negative Selection and Efficient Cre-loxP Mediated Marker Recycling.

In filamentous fungi such as Fusarium graminearum, disruption of multiple genes of interest in the same strain (e.g., to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre-loxP system. The fusion genes contain commonly used C-terminal drug resistance markers, either nptII (G418), nat1 (nourseothricin), or hph (hygromycin B). These resistance genes are fused to the sequence encoding Herpes simplex virus thymidine kinase (HSVtk). Despite the presence of the 1 kb HSVtk gene (about ∼30% increase in total marker size), there is only a slight reduction in transformation efficiency on a molar basis. The fusion genes expressed under the Trichoderma pyruvate kinase (PKI) promoter also confer antibiotic resistance in Escherichia coli, allowing straightforward construction of disruption plasmids. For removal of the loxP flanked resistance cassettes, protoplasts of transformants are directly treated with purified Cre recombinase protein. Loss of the HSVtk containing cassette is selected by restoration of resistance to 5-fluoro-2-deoxyuridine (FdU). As a proof of principle, we demonstrated the efficiency of the HSVtk-based marker removal in Fusarium by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the FgPKS12 gene via integration of the loxP-flanked HSVtk-nptII cassette into the promoter or the first intron, thereby generating transformants with a white mycelium phenotype. Using Cre recombinase and FdU, the selection marker was subsequently removed, and the resulting transformants regained red pigmentation despite the remaining loxP site. We also found that it is possible to remove several unselected loxP-flanked cassettes with a single Cre protein treatment, as long as one of them contains a negative selectable HSVtk cassette. The negative selection system can also be used to introduce allele swaps into strains without leaving marker sequences, by first disrupting the gene of interest and then complementing the deletion in situ with genomic DNA containing a different allele.

Cyanine Photocages Enable Spatial Control of Inducible Cre-Mediated Recombination.

Optical control over protein expression could provide a means to interrogate a range of biological processes. One approach has employed caged ligands of the estrogen receptor (ER) in combination with broadly used ligand-dependent Cre recombinase proteins. Existing approaches use UV or blue wavelengths, which hinders their application in tissue settings. Additionally, issues of payload diffusion can impede fine spatial control over the recombination process. Here, we detail the chemical optimization of a near-infrared (NIR) light-activated variant of the ER antagonist cyclofen. These studies resulted in modification of both the caging group and payload with lipophilic n-butyl esters. The appendage of esters to the cyanine cage improved cellular uptake and retention. The installation of a 4-piperidyl ester enabled high spatial resolution of the light-initiated Cre-mediated recombination event. These studies described chemical modifications with potential general utility for improving spatial control of intracellular caging strategies. Additionally, these efforts will enable future applications to use these molecules in complex physiological settings.

Cell-penetrating peptide-driven Cre recombination in porcine primary cells and generation of marker-free pigs.

Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both in vitro and in vivo. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway. The three CPP–Cre proteins can enter PFFs and subsequently perform recombination with different efficiencies. The recombination efficacy of CPP5–Cre was found to be nearly 90%. The rate-limiting step for CPP–Cre-mediated recombination was the step of endosome escape. HA2 and chloroquine were found to improve the recombination efficiency of TAT–Cre. Furthermore, we successfully obtained marker-free transgenic pigs using TAT–Cre and CPP5–Cre. We provide a framework for the development of CPP-based farm animal transgenic technologies that would be beneficial to agriculture and biomedicine.

Patched1 patterns Fibroblast growth factor 10 and Forkhead box F1 expression during pulmonary branch formation

Hedgehog (Hh) signalling, Fibroblast growth factor 10 (Fgf10) and Forkhead box F1 (Foxf1) are each individually important for directing pulmonary branch formation but their interactions are not well understood. Here we demonstrate that Hh signalling is vital in regulating Foxf1 and Fgf10 expression during branching. The Hedgehog receptor Patched1 (Ptch1) was conditionally inactivated in the lung mesenchyme by Dermo1-Cre in vivo or using a recombinant Cre recombinase protein (HNCre) in lung cultures resulting in cell autonomous activation of Hh signalling. Homozygous mesenchymal Ptch1 deleted embryos (Dermo1Cre+/−;Ptch1lox/lox) showed secondary branching and lobe formation defects. Fgf10 expression is spatially reduced in the distal tip of Dermo1Cre+/−;Ptch1lox/lox lungs and addition of Fgf10 recombinant protein to these lungs in culture has shown partial restoration of branching, indicating Ptch1 function patterns Fgf10 to direct lung branching. Foxf1 expression is upregulated in Dermo1Cre+/−;Ptch1lox/lox lungs, suggesting Foxf1 may mediate Hh signalling effects in the lung mesenchyme. In vitro HNCre-mediated Ptch1 deleted lung explants support the in vivo observations, with evidence of mesenchyme hyperproliferation and this is consistent with the previously reported role of Hh signalling in maintaining mesenchymal cell survival. Consequently it is concluded that during early pseudoglandular stage of lung development Ptch1 patterns Fgf10 and regulates Foxf1 expression in the lung mesenchyme to direct branch formation and this is essential for proper lobe formation and lung function.

Nanoparticle-Mediated Recombinase Delivery into Maize.

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.

Immunohistochemical Procedures for Characterizing the Retinal Expression Patterns of Cre Driver Mouse Lines.

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.

Injection-Based Delivery of Cell-Permeable Peptide-Tagged Cre.

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.

Marker Removal in Transgenic Plants Using Cre Recombinase Delivered with Potato Virus X.

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.

Visualisation of Kiss1 Neurone Distribution Using a Kiss1‐CRE Transgenic Mouse

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE‐GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss‐CRE heterozygous mice with CRE‐activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80‐90% of tdTomato positive neurones in the ARC were co‐labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato‐labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre‐optic areas and to the lateral hypothalamus.