Stereotaxic gene delivery in the rodent brain

Stereotaxic surgery has been an invaluable tool in systems neuroscience, applied in many experiments for the creation of site-targeted lesions, injection of anatomical tracers or implantation of electrodes or microdialysis probes. In this protocol, we describe stereotaxic surgery optimized for gene delivery by recombinant adeno-associated viruses and lentiviruses in mice and rats. This method allows the manipulation of gene expression in the rodent brain with excellent spatiotemporal control; essentially any brain region of choice can be targeted and cells (or a subpopulation of cells) in that region can be stably genetically altered at any postnatal developmental stage up to adulthood. Many aspects of the method, its versatility, ease of application and high reproducibility, make it an attractive approach for studying genetic, cellular and circuit functions in the brain. The entire protocol can be completed in 1-2 hours.     

In vivo DNA electrotransfer into muscle

Naked plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed. Among the non-viral techniques for gene transfer in vivo , this method is especially simple, inexpensive, and safe. However, the relatively low expression levels attained by this method have limited its applications for uses other than as a DNA vaccine. We and other groups investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA vector. The results demonstrated that gene transfer into muscle by in vivo electroporation is far more efficient than simple intramuscular DNA injection and provides a potential approach to systemically delivering cytokines, growth factors, and other serum proteins for basic research and human gene therapy.     

Conditions affecting direct gene transfer into rodent muscle in vivo.

This report extends our previous findings that mouse muscle cells in situ can take up naked DNA injected intramuscularly in vivo. Various conditions such as needle type, speed of injection, volume of injection fluid, tonicity of injection fluid, type of solute, type of muscle, physiologic condition of the muscle and age of the animals were appraised for their effect on the levels of luciferase activity expressed from the pRSVL plasmid. Specific conditions such as the use of normal saline as an injection fluid increased the efficiency of expression. The implantation of DNA pellets was an effective way to deliver DNA to muscle, especially for smaller muscle groups. Also, newborn and adult rat muscles expressed plasmid DNA delivered intramuscularly.     

Insufficient APC capacities of dendritic cells in gene gun-mediated DNA vaccination

Gene gun-mediated DNA immunization is a powerful mode of vaccination against infectious diseases and tumors. Many studies have identified dendritic cells (DC) as the central players in inducing immunity upon biolistic DNA vaccination; however, none of these studies directly quantify DC-mediated responses in comparison with immunity triggered by all Ag- and MHC-expressing cells. In this study we use two different approaches to decipher the relative role of DC vs other cell types in gene gun-induced immunity. First, we directly compared the immunization efficacy of different DNA constructs, which allow Ag expression ubiquitously (CMV promoter) or specifically in DC (CD11c promoter) and would encode either for soluble or membrane bound forms of Ag. Second, we immunized transgenic mice in which only DC can present MHC-restricted Ag, and directly compared the magnitudes of CTL activation with those obtained in wild-type mice. Surprisingly, our combined data suggest that, although DC-specific Ag expression is sufficient to induce humoral responses, DC alone cannot trigger optimal CD4 and CD8 T cell responses upon gene gun vaccination. Therefore, we conclude that DC alone are insufficient to mediate optimal induction of T cell immunity upon gene gun DNA vaccination and that broad Ag expression rather than DC-restricted approaches are necessary for induction of complete immune responses.     

In vitro and in vivo gene delivery by recombinant baculoviruses

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.     

In vivo gene targeting of IL-3 into immature hematopoietic cells through CD117 receptor mediated antibody gene delivery

BACKGROUND: Targeted gene transfection remains a crucial issue to permit the real development of genetic therapy. As such, in vivo targeted transfection of specific subsets of hematopoietic stem cells might help to sustain hematopoietic recovery from bone marrow aplasia by providing local production of growth factors. METHODS: Balb/C mice were injected intravenously, with an anti-mouse c-kit (CD117) monoclonal antibody chemically coupled to a human IL-3 gene-containing plasmid DNA. Mice were sacrificed for tissue analyses at various days after injection of the conjugates. RESULTS: By ELISA, the production of human IL-3 was evidenced in the sera of animals 5 days after treatment. Cytofluorometric analysis after in vivo transfection of a reporter gene eGFP demonstrated transfection of CD117+/Sca1+ hematopoietic immature cells. By PCR analysis of genomic DNA and RNA using primer specific pIL3 sequences, presence and expression of the human IL-3-transgene were detected in the bone marrow up to 10 days in transfected mice but not in control animals. CONCLUSIONS: These data clearly indicate that antibody-mediated endocytosis gene transfer allows the expression of the IL-3 transgene into hematopoietic immature cells, in vivo. While availability of marketed recombinant growth factors is restricted, this targeting strategy should permit delivery of therapeutic genes to tissues of interest through systemic delivery. In particular, the ability to specifically target growth factor expression into repopulating hematopoietic stem cells may create new opportunities for the treatment of primary or radiation-induced marrow failures.     

In vivo electroporation mediated gene delivery to the beating heart

Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase) and TNT (Troponin T) were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.     

In vivo gene delivery and expression by bacteriophage lambda vectors

AIMS: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. METHODS AND RESULTS: Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. CONCLUSIONS: Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.     

Direct DNA delivery into zebrafish embryos employing tissue culture techniques

Summary: The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24–48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources. genesis 31:1–5, 2001. © 2001 Wiley‐Liss, Inc.     

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate.

To target disseminated tumors in vivo, transgenes [beta-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf-beta-galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immune-deficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

siRNA的体内递送

siRNA是一种由siRNA介导的转录后基因沉默。自利用RNAi沉默目的基因获得成功以来, 体内应用RNAi的研究受到高度重视。由于siRNA本身的不稳定性以及体内的复杂环境, siRNA递送的安全性与有效性成为目前关注的重点。文章就目前报道的siRNA体内递送方式进行了综述。     

Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation

We describe the use of direct injection of circular plasmid DNA and subsequent in vivo electroporation (EP) for efficient gene delivery to the ovarian cells, including follicular cells and oocytes of mice. When Trypan blue (TB) was injected into the central portion of an ovary by a glass micropipette, rapid dispersion of TB to each preantral and antral follicle was observed. Injections of lacZ-expressing plasmid DNA and subsequent in vivo EP resulted in transfection of follicles with efficiencies ranging from 8-60%, together with cells in the thecal portion of the ovary. Of the lacZ-positive follicles, some oocytes were also positive for lacZ activity. These findings suggest that a solution introduced inside the ovary is rapidly dispersed to each follicle. With this technique, we expect great progress in genetic engineering in murine ovary.     

In vivo cloning of DNA into multicopy cosmids by mini-Mu-cosduction

Summary A general in vivo procedure for cloning Escherichia coli genes into cosmids has been developed. The method we describe here uses a deleted Mu phage (a mini-Mu) to transpose E. coli genes into cosmids during mini-Mu replication. The resulting cosmids clones are packaged in-vivo into phage particles. Plasmids carrying a particular DNA sequence can be selectively recovered after infection of a new host with the in vivo constructed genomic cosmid library. This system was used succesfully to clone several E. coli genes.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, September 6, 1986     

Targeted gene delivery in the cricket brain,using in vivo electroporation

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb′-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.     

In vivo electroporation: a new frontier for gene delivery and embryology

One of the key techniques in developmental biology is introducing transgenes into tissues and analyzing their subsequent effects on morphogenesis and organogenesis. In mammals, the transgenic approach is a way to misexpress foreign genes in various tissues and organs. However, targeting expression to certain tissues is totally dependent on the availability of specific promoters. Hence, it is not an easy task to control transgene expression temporally and spatially during embryogenesis. Further, if the transgene is toxic, embryonic development can be disrupted, resulting in premature death before the desired stages of development. As alternative systems, Xenopus and zebrafish are used frequently. In these vertebrate models, overexpression of genes can be carried out by injecting synthetic RNAs into eggs. However, genetic techniques in these systems are limited only to early development, prohibiting the precise analysis of gene effects on organogenesis in later stages. In contrast, the chick embryo has long served as a powerful and useful model system, holding a unique position in the field of developmental biology. Although trials of transgenic chicks have never been successful, easy accessibility to the developing embryo through a window opened in an eggshell enables performance of a variety of techniques, such as time-lapse cinephotomatography, microsurgical manipulations (including chick/quail chimeras), transplantation of cells and tissues, New's in vitro culture, etc. (Bortier et al., 1996; Douarin et al., 1996; Selleck, 1996). In addition to these experimental advantages, retrovirus-mediated gene delivery, and recently, adenovirus-mediated misexpression have been employed routinely in chick embryos (Leber et al., 1996; Morgan and Fekete, 1996).