Direct DNA Injection Research Articles

Applications in in ovo technology

By mid-August 1995, 55% of broiler embryos in North America were vaccinated for Marek's disease using the INOVOJECT system, with 201 INOVOJECT machines placed with 16 of the top 25 poultry producers, providing the industry with the capacity to inject in excess of 400 million eggs per month or about 5 billion eggs per annum. In ovo administration of a bursal disease antibody-infectious bursal disease virus (BDA-IBDV) complexed vaccine to specific-pathogen-free (SPF) embryos was safer and more potent than conventional IBDV vaccine alone because it delayed the appearance of bursal lesions, produced no early mortality, produced higher geometric mean antibody titers against IBDV, and generated protective immunity against challenge. In ovo administration of a BDA-IBDV complexed vaccine to broiler embryos generated antibody titers against IBDV sooner than conventional virus vaccinates, and generated protective immunity against challenge Direct DNA injection of plasmid DNA encoding beta-galactosidase into breast muscle in ovo and posthatch was an effective means to achieve both gene transfer and expression, with potential for the development of gene vaccines using plasmids encoding protective antigens from poultry pathogens. In ovo administration of 800 U chicken myelomonocytic growth factor (cMGF), a chicken hematopoietic cytokine for cells of the monocytic-granulocytic lineages, significantly reduced mortality associated with Escherichia coli exposure within the hatcher when compared to PBS controls (6.1 vs 12.4, P < or = 0.05), but not when compared to a yeast expression control. A procedure was developed enabling injection prior to the onset of incubation without compromising embryo viability. This in ovo injection process has opened up the window of embryo development during incubation for intervention, as illustrated by the 100% male phenotype produced in chicks hatching from eggs injected with aromatase inhibitor prior to incubation. These data illustrate some of the in ovo applications presently in use by the poultry industry, and under development or in research at EMBREX.

Use of gonadal primordial germ cells (PGCs) as tools for gene transfer in chickens.

As a result of morphological and developmental characteristics of the early avian embryo, several approaches, taking place at early stages, have been investigated to introduce foreign DNA into the avian genome. They include: 1. Insemination with sperm cells to which foreign DNA was coated (,); 2. Transfection of DNA into follicles before fertilization (), 3. Direct injection of naked DNA into newly fertilized ova or into germinal disk (, , , ), 4. Microinjection of replication-defective retroviral vectors (-) into unincubated embryo blastoderm, and; 5. Retroviral infection of primordial germ cells, PGCs (,).

Chapter 20 DNA- and Adenovirus-Mediated Gene Transfer into Cardiac Muscle

This chapter discusses and compares two methods of gene transfer into the rodent heart: direct plasmid DNA injection and injection of recombinant adenovirus. The predominant use of direct injection into the heart has been to analyze promoter elements in vivo using reporter genes driven by the promoter element of interest. The second general use of in vivo gene transfer is to examine the consequences of expression of a given gene on the structure or function of the heart. This could involve expression of a mutant protein or ectopic expression of an isoform of a protein normally found in another setting. For these purposes DNA injection would not be efficient enough for most forms of phenotypic modification. Therefore, focus has shifted on the use of recombinant adenoviruses. Adenoviruses efficiently infect a large number of replicating and nonreplicating cell types. Their ability to infect nonreplicating cells makes them ideal for use in the heart, because adult cardiac myocytes are terminally differentiated.

Induction of HIV-1 Nef-specific cytotoxic T lymphocytes by Nef-expressing DNA vaccine.

Recently, some individuals who have remained seronegative despite definite exposure to HIV-1 have been reported. Among these individuals, an unusually high frequency of HIV-1 Nef-specific cytotoxic T lymphocytes was observed. Direct injection of plasmid DNA encoding foreign antigen can elicit both cell-mediated immunity and antibody responses (DNA vaccine). We constructed an HIV-1 Nef-expressing plasmid, and we induced HIV-1 Nef-specific cytotoxic T lymphocytes. This is the first report of inducing HIV-1 Nef-specific cytotoxic T lymphocytes by DNA vaccine.

Induction of HIV‐1 Nef‐specific cytotoxic T lymphocytes by Nef‐expressing DNA vaccine

Recently, some individuals who have remained seronegative despite definite exposure to HIV-1 have been reported. Among these individuals, an unusually high frequency of HIV-1 Nef-specific cytotoxic T lymphocytes was observed. Direct injection of plasmid DNA encoding foreign antigen can elicit both cell-mediated immunity and antibody responses (DNA vaccine). We constructed an HIV-1 Nef-expressing plasmid, and we induced HIV-1 Nef-specific cytotoxic T lymphocytes. This is the first report of inducing HIV-1 Nef-specific cytotoxic T lymphocytes by DNA vaccine. © 1996 Wiley-Liss, Inc.

Phenobarbital Induction Mediated by a Distal CYP2B2 Sequence in Rat Liver Transiently Transfected in Situ

The promoter activities of the genes for cytochrome P450 2B1 (CYP2B1) and cytochrome P450 2C1 (CYP2C1) have been assayed by direct injection of promoter-luciferase chimeric genes into rat liver. Activities of minimal promoters for CYP2C1 and CYP2B1 were detectable in untreated animals but were not increased by treatment of the animals with phenobarbital. After insertion to the 5' side of the minimal promoters of one to three copies of the CYP2B2 sequence from -2318 to -2155, a phenobarbital-responsive element in primary hepatocyte cultures (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene (Amst.) 158, 263-268), phenobarbital treatment induced the activity of the CYP2C1 promoter by 5-15-fold and the CYP2B1 promoter by 2.5-5-fold. Mutation of a basal transcription element-like motif and a CCAAT/enhancer binding protein element in the CYP2B1 proximal promoter region reduced expression, but 3-4-fold induction by phenobarbital was retained. Mutation of the "Barbie box," a putative phenobarbital-responsive element (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869) in the CYP2B1 proximal promoter did not reduce the relative response to phenobarbital. These results demonstrate that direct injection of DNA into rat liver may be used to assay phenobarbital responsiveness of cytochrome P450 genes. In this system, a distal CYP2B2 element mediates a response to phenobarbital, and proximal elements, including the Barbie box, are not required for the induction.

Identification and Characterization of a 47 Base Pair Activity-Dependent Enhancer of the Rat Nicotinic Acetylcholine Receptor δ-Subunit Promoter

Nicotinic acetylcholine receptor (nAChR) genes are regulated by muscle electrical activity. E-box sequences found in their promoters are necessary for this regulation. However, many muscle genes contain E-boxes, yet are not regulated by muscle depolarization. This suggests that other elements are necessary, perhaps working in conjunction with E-boxes, to confer depolarization-dependent control onto promoter activity. We have used direct DNA injection into muscle as an in vivo assay to identify and characterize these additional elements. Mutagenesis and expression assays identified multiple elements within the first 81 base pairs (bp) of the nAChR delta-subunit promoter that contribute to its regulation by muscle electrical activity. Within this 81 bp sequence, two regions of DNA were identified that were capable of conferring activity-dependent regulation onto a heterologous promoter. The stronger of these two putative enhancers was characterized further. It is a 47 bp sequence that contains an E-box along with sequences similar to the SV40 core enhancer and an SP1 site. Site-directed mutagenesis identified residues within each of these sequences that were necessary for enhancer activity. Furthermore, methylation interference DNA footprinting assays showed increased nuclear protein binding to sequences within both these enhancers after muscle denervation, and this pattern of binding was very similar to that observed with nuclear protein isolated from myotube extracts. These latter results suggest that similar mechanisms may mediate increased nAChR expression during muscle development and after muscle denervation.

Expression of exogenous genes trasnferred into the avian limb in ovo

We report on a simple method of direct gene transfer which allows the ectopic expression of proteins and the study of mesoderm-specific genes in the chick embryo. We microinjected into the avian embryonic limb several plasmids containing reporter genes under the control of various promoter sequences, including a minimal chicken muscle acetylcholine receptor α-subunit promoter [Klarsfeld, A., Daubas, A., Bourachot, B. and Changeux, J.P., Mol. Cell. Biol., 7 (1987) 951–955]. Gene expression is detectable for 3 days, is reproducible, is restricted to the site of injection, and correlates with the amount of DNA injected. Our observations indicate that it is possible to transfer and express genes in ectodermal and mesodermal cells of the chick limb by direct DNA injection and that the method can be used to analyze promoter sequences in vivo during specific windows of development.

Gene expression and active virus replication in the liver after injection of duck hepatitis B virus DNA into the peripheral vein of ducklings

Background/Aims: Duck hepatitis B virus is a member of the hepadnavirus family, which posses strong hepatotropism. Duck hepatitis B virus DNA serves as the replicative template for producing biologically active virus particles after transfection into cell lines established from human hepatocellular carcinoma or into duck liver by direct injection of calcium phosphate-precipitated DNA. Our aim was to develop a new method of liver-specific gene expression after intravenous DNA delivery. Methods/Results: We inoculated duck hepatitis B virus DNA with and without cationic liposomers, Lipofectin or LipofectAMINE, as DNA carriers. Two weeks after a single intravenous injection of 10 or 50 μg of plasmid DNA containing a head-to-tail dimer of duck hepatitis B virus DNA into 25 one-day old ducklings, duck hepatitis B virus RNA transcripts including the pregenome replicative intermediate were detected by Northern blot in the liver of eight ducks (100%) of the Lipofectin group, five ducks (63%) of the LipofectAMINE group, and three ducks (50%) of the group which received DNA without carrier. Duck hepatitis B virus RNA transcription was almost exclusively liver specific, even though the lipsomes had no tissue specificity. Replicative forms of duck hepatitis B virus DNA were detected in the liver and DHBsAg was observed in the cytoplasm of the hepatocytes by immunostaining. The serum of transfected ducklings contained virus particles which were infectious in other ducklings. Conclusion: The efficient and liver-specific expression of inoculated DNA was due to the amplification of nucleic acids by active virus replication process under the control of hepatocyte specific regulation.

Successful gene therapy following direct injection of naked DNA encoding secreted protein into skeletal muscle

Successful gene therapy following direct injection of naked DNA encoding secreted protein into skeletal muscle

Gene therapy of Parkinson disease model rat by direct injection of plasmid DNA-lipofectin complex.

Lipofectin-mediated gene transfer was used to introduce plasmid harboring the tyrosine hydroxylase (TH) gene into the striatum of rats with lesions of the nigrostriatal pathway. The rotational asymmetry of Parkinson disease model rat was reduced quickly and significantly, suggesting that plasmid-DNA-transfected brain cells can generate L-dopa locally in the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input. Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) also confirm that striatal cells can express exogenous TH gene. Such in vivo plasmid DNA transfer strategy may be useful in other neurologic disease therapy, especially acute brain insults.

Cellular and Molecular Barriers to Gene Transfer by a Cationic Lipid

Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.

Cytokine gene expression in epidermis with biological effects following injection of naked DNA.

The epidermis is readily accessible for genetic manipulation and is easily monitored. Using pig skin because it is very similar to human skin morphologically, we have developed a method to transiently express biologically active factors in epidermis. Following direct injection of naked plasmid DNA into skin, DNA is taken up and transiently expressed at high levels by epidermal keratinocytes. Injection of interleukin-8 plasmid DNA into skin results in the appropriate biological response of neutrophil recruitment, demonstrating functional utility. In addition to this model's therapeutic uses, the biological effects of structural gene products on the epidermis could also be studied in vivo.

Intramuscular injection of plasmid DNA.

Striated muscle has recently been shown to be an excellent target tissue for gene transfer in vivo. Both skeletal and cardiac muscle are post-mitotic highly stable structures; consequently gene transfer into these tissues produces long-lasting gene expression. A number of gene transfer systems work effectively with skeletal muscle, including retroviral gene vectors (Dunckley et al., 1993) and adenoviral vectors (Ragot et al., 1993) but the simplest method is the direct injection of naked plasmid DNA into the muscle. Gene uptake and expression following in vivo transfection with plasmid DNA is substantially higher in striated muscle compared with any other tissue (Acsadi et al., 1991a). This methodology, first reported by Wolff and coworkers (1990), has attracted considerable interest as a means of in vivo gene therapy for a wide range of diseases. Skeletal muscle can serve as a platform for systemic gene expression; for example the production of Factor VIII for the treatment of hemophilia A, as well as being the target for treatment, as in the case of Duchenne muscular dystrophy. Plasmid DNA can also be used as a means of delivering genetic vaccination, an approach that potentially can give rise to effective long-lasting humoral and cell-mediated immunity following a single treatment (see Chapter 18).

Intramuscular delivery of rat kallikrein-binding protein gene reverses hypotension in transgenic mice expressing human tissue kallikrein.

The tissue kallikrein-kinin system has been postulated to play a role in blood pressure regulation. The activity of tissue kallikrein is controlled by a number of factors in vivo. Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor which binds to and inhibits tissue kallikrein's activity in vitro. We have recently developed several hypotensive transgenic mouse lines which express human tissue kallikrein. In order to investigate the role of RKBP in blood pressure regulation, we delivered the RKBP to these transgenic mice by intramuscular injection. Expression of the RKBP was detected in skeletal muscle by reverse transcription-polymerase chain reaction and Southern blot analysis at 10, 20, 30, and 40 days post-injection. Immunoreactive RKBP levels in the muscle and serum of these mice were quantified by a RKBP-specific enzyme-linked immunosorbent assay and Western blot analysis. The levels of RKBP mRNA and immunoreactive protein were detectable at 10 days post-injection and increased significantly at 20 and 30 days. During this period, RKBP delivery significantly increased systemic blood pressure in the kallikrein transgenic mice to a level comparable to that of normotensive control mice. The RKBP and vector DNA delivery had no effect on the blood pressure of normotensive control mice. No serum antibodies to RKBP or its DNA were detected in the mice 40 days post injection. These results suggest that the increase of systemic blood pressure by RKBP delivery in these hypotensive transgenic mice may be mediated by inhibiting tissue kallikrein activity.

Gene expression following direct injection of DNA into liver.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Dexamethasone enhancement of gene expression after direct hepatic DNA injection.

The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene delivery. This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats. Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal repeat (RSV LTR). Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression. Southern analysis of total DNA extracted from liver at various times following injection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern. This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.

Tissue Restricted Gene Expression Assayed by Direct DNA Injection into Cardiac and Skeletal Muscle

We have used direct injection of plasmid DNA into heart and tongue in vivo and transfection into cells in culture to determine (1) whether the pattern of reporter gene expression parallels the cell-type specific expression of endogenous genes. (2) whether the pattern of reporter gene expression approximates that of the same constructs transfected into myocardiocytes and myogenic cells in culture, (3) whether the expression patterns of promoters and enhancers that had been subtly altered by mutations are similar following transfection and direct DNA injection. We utilized gene constructs derived from the two fiber-type specific human troponin C genes: the fast twitch gene (TnCf) and the slow twitch or cardiac gene (cTnC). The endogenous TnCf gene does not express in the heart and the plasmid DNA expresses neither in myocardium after injection nor in transfected cardiomyocytes. However injected TnCf does express vigorously in tongue muscle. Conversely, the endogenous cTnC gene does not express in fast twitch skeletal muscle tongue and the plasmid DNA does not express in tongue after injection. However, injected cTnC does express in both injected myocardium and in transfected cardiomyocytes. With few discrepancies, various deletions and alterations of the promoters and enhances of these genes that have previously been defined by transfection into permissive myogenic cells in culture gave parallel expression patterns after injection in myocardium and tongue. Thus we conclude that direct DNA injection appears to provide a method to verify the identities of important cis-acting regulatory regions that have been mapped in cells in culture.

In VivoGene Transfer into Rabbit Thyroid Follicular Cells by Direct DNA Injection

Direct injection of DNA expression vectors into muscle leads to expression of encoded recombinant gene products in mature muscle cells. This phenomenon is not shared by most other organs. We have surveyed various organs in the rabbit to identify other cell types that would express DNA vectors after direct injection. We observed that thyroid follicular cells were capable of acquiring plasmid DNA and expressing recombinant gene products after direct interstitial injection of plasmid vectors into the thyroid gland. The level of expression of a chloramphenicol acetyltransferase (CAT) reporter gene in thyroid tissue was similar to that seen in muscle tissue three days after injection in controlled experiments. Using a beta-galactosidase reporter gene, expression was localized to thyroid follicular cells. CAT activity decreased with first-order kinetics and a half-life t1/2 of 40 hr. DNA was identified in thyroid tissue by polymerase chain reaction (PCR) analysis and displayed first-order elimination kinetics with a half-life t1/2 of 10 hr. The persistence of the gene and gene product in the thyroid was significantly different from that observed after injection of DNA vectors into muscle or delivery of DNA vectors to the liver using asialoglycoprotein/polylysine/DNA complexes, suggesting that there are significant differences in the process of DNA uptake or compartmentalization in these experimental systems. These results introduce the possibility of developing the thyroid as a novel target for treating certain thyroid or systemic diseases using DNA vectors.

Transgenic mice: a decade of progress in technology and research

In little more than a decade, the techniques developed for altering the genetic makeup of laboratory and livestock animals and plants have changed the landscape of biological research. It is now possible to introduce virtually any cloned gene into the germ line and study the expression pattern and pattern and effects of the inroduced gene, or transgene. This has allowed the extension of in vitro and in vivo cell-culture studies into whole animal systems in which the introduced gene is subjected to all normal regulatory processes from the onset of development. Although there have been reports of foreign gene expression resulting from direct injection of DNA in animals (e.g., Wolff et al., 1990; Zhu et al., 1993), transgenic animals are the primary models for examining molecular genetic phenomena in vivo.