ABSTRACT
Genome-editing tools such as Cre recombinase (Cre), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biomedical research, agriculture, microbial engineering, and therapeutic development. Direct delivery of genome editing enzymes, as opposed to their corresponding DNA and mRNA precursors, is advantageous since they do not require transcription and/or translation. In addition, prolonged overexpression is a problem when delivering viral vector or plasmid DNA which is bypassed when delivering whole proteins. This lowers the risk of insertional mutagenesis and makes for relatively easier manufacturing. However, a major limitation of utilizing genome editing proteins in vivo is their low delivery efficiency, and currently the most successful strategy involves using potentially immunogenic viral vectors. This lack of safe and effective non-viral delivery systems is still a big hurdle for the clinical translation of such enzymes. This review discusses the challenges of non-viral delivery strategies of widely used genome editing enzymes, including Cre recombinase, ZFNs and TALENs, CRISPR/Cas9, and Cas12a (Cpf1) in their protein format and highlights recent innovations of non-viral delivery strategies which have the potential to overcome current delivery limitations and advance the clinical translation of genome editing.
Subject(s)
Gene Editing/methods , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Bacterial Proteins/administration & dosage , CRISPR-Associated Proteins/administration & dosage , Clustered Regularly Interspaced Short Palindromic Repeats , Dendrimers/chemistry , Endodeoxyribonucleases/administration & dosage , Gold/chemistry , Integrases/administration & dosage , Lipids/chemistry , Nanoparticles/chemistry , Phosphorus/chemistry , Polyethyleneimine/chemistry , Transcription Activator-Like Effector Nucleases/administration & dosage , Zinc Finger Nucleases/administration & dosageABSTRACT
The scarcity of effective means to deliver functional proteins to living cells is a central problem in biotechnology and medicine. Herein, we report the efficient delivery of an active DNA-modifying enzyme to human stem cells through high-density cell penetrating peptide brush polymers. Cre recombinase is mixed with a fluorophore-tagged polymer carrier and then applied directly to induced pluripotent stem cells or HEK293T cells. This results in efficient delivery of Cre protein as measured by activation of a genomically integrated Cre-mediated recombination reporter. We observed that brush polymer formulations utilizing cell penetrating peptides promoted Cre delivery but oligopeptides alone or oligopeptides displayed on nanoparticles did not. Overall, we report the efficient delivery of a genome-modifying enzyme to stem cells that may be generalizable to other, difficult-to-transduce cell types.
Subject(s)
Drug Delivery Systems , Gene Editing , Induced Pluripotent Stem Cells/metabolism , Integrases/administration & dosage , Integrases/metabolism , Peptides/chemistry , Polymers/chemistry , HEK293 Cells , Humans , Molecular Structure , Peptides/pharmacokinetics , Polymers/pharmacokineticsABSTRACT
Cre/loxP recombination is a widely used approach to study gene function in vivo, using mice models expressing the Cre recombinase under the control of specific promoters or through viral delivery of Cre-expressing constructs. A profuse literature on transgenic mouse lines points out the deleterious effects of Cre expression in various cell types and tissues, presumably by acting on illegitimate loxP-like sites present in the genome. However, most studies reporting the consequences of Cre-lox gene invalidation often omit adequate controls to exclude the potential toxic effects of Cre, compromising the interpretation of data. In this study, we report the anatomical, neurochemical, and behavioral consequences in mice of adeno-associated virus (AAV)-mediated Cre expression in the dopaminergic nuclei substantia nigra, at commonly used viral titers (3 × 109 genome copies/0.3 µL or 2 × 109 genome copies/0.6 µL). We found that injecting AAV-eGFP-Cre into the SN engendered drastic and reproducible modifications of behavior, with increased basal locomotor activity as well as impaired locomotor response to cocaine compared to AAV-eGFP-injected controls. Cre expression in the SN induced a massive decrease in neuronal populations of both pars compacta and pars reticulata and dopamine depletion in the nigrostriatal pathway. This anatomical injury was associated with typical features of programmed cell death, including an increase in DNA break markers, evidence of apoptosis, and disrupted macroautophagy. These observations underscore the need for careful control of Cre toxicity in the brain and the reassessment of previous studies. In addition, our findings suggest that Cre-mediated ablation may constitute an efficient tool to explore the function of specific cell populations and areas in the brain, and the impact of neurodegeneration in these populations.
Subject(s)
Integrases , Neurons/pathology , Substantia Nigra/metabolism , Substantia Nigra/pathology , Animals , Apoptosis/drug effects , Dependovirus , Dopamine/metabolism , Genetic Vectors , Integrases/administration & dosage , Integrases/genetics , Integrases/toxicity , Locomotion/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/drug effects , Neurons/metabolismABSTRACT
Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.
Subject(s)
Cerebral Cortex/physiology , Electroporation/methods , Integrases/administration & dosage , Integrases/metabolism , Animals , Integrases/genetics , Mice , Recombination, GeneticABSTRACT
Genetic mouse models of soft tissue sarcoma provide critical insights into disease pathophysiology, which are oftentimes unable to be extracted from human tumor samples or xenograft models. In this study we describe a mouse model of soft tissue sarcoma mediated by adenoviral-Cre recombinase injection into Trp53fl/fl/Ptenfl/fl lox-stop-lox luciferase mice. Injection of adenovirus expressing Cre recombinase, either subcutaneously or intramuscularly in two experimental groups, results in viral infection and gene recombination with 100% penetrance within the first 24 hours following injection. Luciferase expression measured by real-time bioluminescence imaging increases over time, with an initial robust increase following viral injection, followed by a steady rise over the next several weeks as primary tumors develop and grow. Intramuscular injections were more commonly associated with evidence of systemic viral distribution than subcutaneous injections. All mice developed soft tissue sarcomas at the primary injection site, with histological examination identifying 93% of tumors as invasive pleomorphic sarcomas based on microscopic morphology and immunohistochemical expression of sarcoma markers. A lymphocytic infiltrate was present in 64% of the sarcomas in this immunocompetent model and 71% of tumors expressed PD-L1. This is the first report of a viral-Cre mediated Trp53/Pten mouse model of undifferentiated pleomorphic sarcoma. The bioluminescence imaging feature, along with high penetrance of the model and its immunological characteristics, makes it suited for pre-clinical studies of soft tissue sarcoma.
Subject(s)
Cell Differentiation , Disease Models, Animal , Integrases/administration & dosage , PTEN Phosphohydrolase/genetics , Sarcoma/pathology , Soft Tissue Neoplasms/pathology , Tumor Suppressor Protein p53/genetics , Animals , Luminescence , Mice , Mice, Inbred C57BL , Sarcoma/genetics , Soft Tissue Neoplasms/geneticsABSTRACT
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.
Subject(s)
Cell-Penetrating Peptides/genetics , Gene Editing/methods , Genome , Integrases/genetics , Recombinant Fusion Proteins/genetics , Recombination, Genetic , Animals , Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Genetic Loci , Hydrogels/administration & dosage , Hydrogels/chemistry , Injections, Intramuscular , Integrases/administration & dosage , Integrases/metabolism , Mice , Muscle, Skeletal/metabolism , Myocardium/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Plasmids/administration & dosage , Plasmids/chemistry , Plasmids/metabolism , Primary Cell Culture , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/metabolism , UltrasonographyABSTRACT
In the present study, we generated novel cre driver mice for gene manipulation in pancreatic ß cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1(em1 (cre) Utr) strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1(em1 (cre) Utr)) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting ß cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1(em1 (cre) Utr) is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.
Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Insulin-Secreting Cells , Insulin/genetics , Integrases/genetics , Mice, Mutant Strains , Animals , Codon, Terminator/genetics , Crk-Associated Substrate Protein/administration & dosage , Glucose/metabolism , Injections , Integrases/administration & dosage , Mice, Inbred C57BL , Mice, Mutant Strains/genetics , Mutagenesis, Insertional , Oocytes , RNA/administration & dosage , Recombination, GeneticABSTRACT
A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.
Subject(s)
Gene Knockout Techniques , Genes, Synthetic , Genetic Engineering/methods , Lipids/chemistry , Nanoparticles , Animals , Bacterial Proteins/administration & dosage , Bacterial Proteins/genetics , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Ceramides/chemistry , Cholesterol/chemistry , Drug Carriers , Endocytosis , Endonucleases/administration & dosage , Endonucleases/genetics , Endosomes/metabolism , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Hypothalamus/metabolism , Integrases/administration & dosage , Integrases/genetics , Lipids/administration & dosage , Lipids/chemical synthesis , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , Molecular Structure , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Nanoparticles/metabolism , Nanoparticles/toxicity , Phosphatidylethanolamines/chemistry , RNA/genetics , Recombinant Proteins/biosynthesis , Recombination, Genetic , Static Electricity , Structure-Activity Relationship , Thalamus/metabolismABSTRACT
GATA4 and GATA6 are central cardiac transcriptional regulators. The postnatal, stage-specific function of the cardiac transcription factors GATA4 and GATA6 have not been evaluated. In part, this is because current Cre-loxP approaches to cardiac gene inactivation require time consuming and costly breeding of Cre-expressing and "floxed" mouse lines, often with limited control of the extent or timing of gene inactivation. We investigated the stage-specific functions of GATA4 and GATA6 in the postnatal heart by using adeno-associated virus serotype 9 to control the timing and extent of gene inactivation by Cre. Systemic delivery of recombinant, adeno-associated virus 9 (AAV9) expressing Cre from the cardiac specific Tnnt2 promoter was well tolerated and selectively and efficiently recombined floxed target genes in cardiomyocytes. AAV9:Tnnt2-Cre efficiently inactivated Gata4 and Gata6. Neonatal Gata4/6 inactivation caused severe, rapidly lethal systolic heart failure. In contrast, Gata4/6 inactivation in adult heart caused only mild systolic dysfunction but severe diastolic dysfunction. Reducing the dose of AAV9:Tnnt2-Cre generated mosaics in which scattered cardiomyocytes lacked Gata4/6. This mosaic knockout revealed that Gata4/6 are required cell autonomously for physiological cardiomyocyte growth. Our results define novel roles of GATA4 and GATA6 in the neonatal and adult heart. Furthermore, our data demonstrate that evaluation of gene function hinges on controlling the timing and extent of gene inactivation. AAV9:Tnnt2-Cre is a powerful tool for controlling these parameters.
Subject(s)
Dependovirus/genetics , GATA4 Transcription Factor/metabolism , GATA6 Transcription Factor/metabolism , Heart/growth & development , Integrases/genetics , Myocytes, Cardiac/physiology , Animals , Animals, Newborn , GATA4 Transcription Factor/genetics , GATA6 Transcription Factor/genetics , Gene Expression Regulation, Developmental , Gene Silencing , Genetic Vectors , Heart/physiology , Heart Failure, Systolic/genetics , Integrases/administration & dosage , Mice, TransgenicABSTRACT
INTRODUCTION: The φC31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells. AREAS COVERED: The φC31 integrase system has been under development for ten years and has been demonstrated to be effective for integration of plasmids in a variety of tissues and organs for gene therapy in animal systems, as well as in isolated human cells. We focus on work with the φC31 integrase system during the past 12-18 months. This work has centered on a series of papers involving in vivo delivery of the integrase system to the liver and a variety of studies demonstrating the utility of the integrase system in stem cells. EXPERT OPINION: We conclude that the φC31 integrase system has significant potential for liver gene therapy, if effective DNA delivery methods for large mammals become available. The φC31 integrase system displays an outstanding fit for use in pluripotent stem cells, and this area is expected to be the subject of intense development.
Subject(s)
Attachment Sites, Microbiological/physiology , Bacteriophages/genetics , Genetic Therapy/methods , Integrases/administration & dosage , Integrases/genetics , Animals , Bacteriophages/enzymology , Binding Sites/physiology , Genetic Therapy/trends , Humans , Integrases/metabolism , Liver Diseases/genetics , Liver Diseases/therapy , Pluripotent Stem Cells/transplantation , Pluripotent Stem Cells/virology , Streptomyces/genetics , Streptomyces/virologyABSTRACT
Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >10¹° infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads.
Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors/chemical synthesis , Helper Viruses/genetics , Integrases/administration & dosage , Doxycycline/pharmacology , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacologyABSTRACT
The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.
Subject(s)
Genes, Reporter , Integrases/administration & dosage , Receptors, CCR2/genetics , Receptors, CCR5/genetics , Zygote , Animals , Base Sequence , DNA Primers , Embryonic Stem Cells/metabolism , Gene Deletion , Genetic Vectors , Germ Cells , In Situ Hybridization, Fluorescence , Integrases/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microinjections , Recombination, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Cre recombinase, when used as a tool in agricultural biotechnology, can precisely excise DNA sequences that may be useful in the introduction of a new trait but are not needed in the commercial product. Although the cre genetic material would not be present in the final product, the present studies were performed to assess the safety of Cre recombinase to provide confirmatory evidence of the safe use of Cre-lox technology in agricultural biotechnology. Cre recombinase shares no relevant sequence similarity to known allergens or toxins. When Cre recombinase was exposed to a pH 1.2 solution of simulated gastric fluid lacking pepsin, CD spectroscopy showed that there was a loss of secondary structure and that the protein was no longer active in a functional assay. Cre recombinase was degraded rapidly when exposed to pepsin in a standardized gastric digestion model; therefore, Cre recombinase would not survive the harsh gastric environment. When orally administered to mice as an acute dosage of 53 mg/kg of body weight, no treatment-related adverse findings were observed. These data support the conclusion that human and animal dietary exposure to Cre recombinase pose no known safety concerns; consistent with the fact that bacteriophage P1, the source of the cre gene and expressed protein, is commonly encountered in the environment and in normal enteric bacteria without reports of adverse consequences.
Subject(s)
Biotechnology/standards , Food, Genetically Modified/standards , Integrases/administration & dosage , Integrases/adverse effects , Acids , Administration, Oral , Amino Acid Sequence , Animals , Body Weight/drug effects , Circular Dichroism , Enzyme Stability , Food, Genetically Modified/adverse effects , Integrases/genetics , Integrases/metabolism , Mice , Molecular Sequence Data , Protein Conformation , Safety , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Cartilage differentiation occurs over a broad time range from early embryonic development, when the mesenchymal condensations that give rise to cartilage models for future bone first appear, and continuing through adult life, when there is ongoing maintenance of articular joint surfaces and re-activation of cartilage formation after fracture. The chondrogenic response also figures in the pathogenesis of degenerative and inflammatory joint diseases. We have generated a transgenic line expressing tamoxifen-dependent Cre recombinase that gives efficient recombination in the chondrogenic lineage, both during embryogenesis and postnatally, and provides a valuable tool for analysis of gene function selectively in chondrogenic cells using conditional genetic approaches. Because the cartilage model of the limb skeleton forms progressively in a proximodistal order during discrete, well-defined time periods, evaluation of the spatial extent of tamoxifen-induced recombination along the limb axis during these time windows has also enabled us to examine the pharmacokinetics of single-dose tamoxifen injections during pregnancy.
Subject(s)
Cartilage/embryology , Cartilage/enzymology , Extremities/embryology , Integrases/metabolism , Tamoxifen/pharmacology , Animals , Animals, Newborn , Base Sequence , Cartilage/drug effects , Cartilage/growth & development , Chondrogenesis/drug effects , Collagen Type II/genetics , DNA Primers/genetics , Extremities/growth & development , Female , Gene Expression Regulation, Developmental/drug effects , Genes, Reporter , Gestational Age , Integrases/administration & dosage , Kinetics , Lac Operon , Mice , Mice, Transgenic , Pregnancy , Recombination, Genetic/drug effects , Tamoxifen/pharmacokineticsABSTRACT
The Sleeping Beauty transposon system (SB) has been shown to mediate nonviral integration of expression constructs resulting in long-term gene expression in several mammalian targets. Often, however, it is difficult to discern long-term expression resulting from transposition vs nonhomologous chromosomal recombination or maintenance of plasmid DNA in an extrachromosomal form. We have designed a system to silence expression from nontransposed sequences, making it possible to determine more specifically the amount of expression resulting from transposition. A transposon plasmid, pT2F/Cage (carrying a murine erythropoietin (Epo) gene transcriptionally regulated by the ubiquitously expressed CAGS promoter), was engineered to contain LoxP sites positioned so as to interrupt expression upon Cre-mediated recombination. Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing. Interferon-inducible Mx1Cre mice were administered pT2F/Cage with or without transposase-encoding plasmid. At 2 to 4 weeks postinjection, in the absence of SB transposase, Cre induction reduced Epo expression to about 1% of that seen in the group that was administered transposase-encoding plasmid, which maintained Epo levels near those of the uninduced groups. Southern hybridization analysis and plasmid rescue of transfected tissue supported the efficient Cre-mediated silencing of nontransposed sequences. These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase. The results also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression.
Subject(s)
DNA Transposable Elements/genetics , Extracellular Matrix Proteins/genetics , Gene Transfer Techniques , Integrases/genetics , Protein-Lysine 6-Oxidase/genetics , Transposases/genetics , Animals , Erythropoietin/genetics , Extracellular Matrix Proteins/administration & dosage , Extracellular Matrix Proteins/biosynthesis , Gene Silencing , Genetic Vectors/administration & dosage , HeLa Cells , Humans , Integrases/administration & dosage , Integrases/biosynthesis , Liver/enzymology , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Protein-Lysine 6-Oxidase/administration & dosage , Protein-Lysine 6-Oxidase/biosynthesis , Transposases/physiologyABSTRACT
BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.
Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Integrases/administration & dosage , Integrases/genetics , Lentivirus/genetics , Neurons/metabolism , Viral Proteins/administration & dosage , Viral Proteins/genetics , Animals , Cell Line , Gene Expression , Genes, Reporter , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Green Fluorescent Proteins , Hippocampus/metabolism , Hippocampus/virology , Integrases/metabolism , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , Mice, Transgenic , Neocortex/metabolism , Neocortex/virology , Neurons/virology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombination, Genetic/drug effects , Recombination, Genetic/genetics , Transgenes , Viral Proteins/metabolismABSTRACT
To determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, the NMDAR1 (NR1) subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein are reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, are reduced 86-88% in lamina II neurons. The spatial NR1 knock-out does not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. Our results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury.