Gene transfer into chicken embryos by retrovirus vectors

While chickens have many properties that are advantageous for embryological studies, their genetic analysis has been restricted. However, by using retrovirus vector systems in combination with classical techniques of experimental developmental biology, it has recently become possible to analyze the function of genes involved in the development of this organism. Avian retrovirus vectors are unique in that they can be divided into two categories: replication-competent and replication-defective (replication-incompetent). By choosing the vectors correctly, there are many experimental applications of these vectors such as induction of constitutive (or regulated) gene expression in a restricted region of tissues, organs and embryos; cell lineage analysis; and formation of concentration gradients of morphogens in micromass cultures. In this paper, several retrovirus vectors available for the chicken will be introduced and their applications in developmental biology will be reviewed.     

Gene transfer into skeletal muscle using novel AAV serotypes

BACKGROUND: Skeletal muscle is an interesting target for gene delivery because of its mass and because the vectors can be delivered in a noninvasive way. Adeno-associated virus (AAV) vectors are capable of transducing skeletal muscle fibers and achieving stable and safe transgene expression. To date, most animal experiments using AAV have been based on AAV serotype 2, but some recent studies have demonstrated that AAV1 is more efficient than AAV2/2 in transducing muscle fibers. Recently, novel AAVs (AAV7 and AAV8) were isolated from rhesus macaques. METHODS: We injected three different muscles (gastrocnemius, soleus, biceps femoris) of immunocompetent C57BL/6 mice with different pseudotyped AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8) and quantitatively compared the different gene transfer efficiencies. RESULTS: The efficiencies of transduction in skeletal muscle with AAV2/7 and AAV2/8 were similar to AAV2/1, and higher than that seen with AAV2/2 and AAV2/5. All serotypes were able to transduce both slow and fast muscle fibers similarly at the vector titer used (1x10(11) genome copies per mouse). Despite a limited inflammatory response (slightly higher when using AAV2/2, AAV2/7 and AAV2/8 vectors than AAV2/1 and AAV2/5), transgene expression was observed throughout the length of the experiment. DISCUSSION: These results show that AAV2/7 and AAV2/8 are able to transduce muscle fibers of immunocompetent mice very efficiently, offering new perspectives in gene transfer of skeletal muscle.     

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges.

The titer of retroviral vectors can be increased by cocultivation of retrovirus packaging cells that produce a vector with packaging cells having a different host range. Multiple rounds of infection occur in such cultures, producing an amplification of vector copy number and titer. Production of a vector with a very high titer of over 10(10) CFU per ml of conditioned medium has been reported, although replication-competent helper virus was also present. Since helper-free virus is a requirement for many applications of retroviral vectors, we repeated this procedure with a modified vector and achieved a 2- to 10-fold amplification of vector titer in the absence of helper virus, up to 2 x 10(7) CFU/ml. We have also repeated these experiments with the same vector and methods described previously or have assayed virus from the high-titer vector-producing cell line reported previously and observed maximum titers of 10(8) CFU/ml, invariably accompanied by helper virus. Thus, while amplification of vector titer in the absence of helper virus is possible, some unexplained difference in the assays for virus titer must account for our inability to obtain the exceptionally high vector titers that were reported previously.     

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.     

Gene transfer into hemopoietic stem cells using retroviral vectors

Gene transfer to hemopoietic cells offers a variety of new approaches to the experimental hematologist as well as potentially providing a means for correcting a number of genetic disorders of humans. From the experimental viewpoint, gene transfer utilizing retroviral vectors introduces new methods for analyzing hemopoietic cell lineages, and the effects of over-expression of genes affecting hemopoietic cell proliferation and differentiation. The unique properties of retroviral vectors and the optimized methods currently in use to infect hemopoietic cells are represented as a brief review of a rapidly expanding new field of experimental hematology.     

Gene transfer mediated by alpha2-macroglobulin.

alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein.     

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus

Summary The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for β-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of β-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were β-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.     

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.     

Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures

Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

Gene transfer to cereal cells mediated by protoplast transformation

Summary Direct gene transfer to cereal cells was achieved by transformation of protoplasts with naked DNA. Protoplasts isolated from cultured cells of Triticum monococcum were incubated in the presence of polyethylene glycol (PEG) with circular and linear plasmid DNA. The pBR322-derived plasmid, pBL1103-4, contained a selectable chimeric gene comprising the protein coding region of the Tn5 aminogly-coside phosphotransferase type II gene (NPT II), the nopaline synthase promoter (pNOS) and the polyadenylation signal of the octopine synthase gene. Transformed cells were selected in medium containing kanamycin and identified by detection of aminoglycoside phosphotransferase II activity.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Gene delivery into hepatocytes with the preS/liposome/DNA system

Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system.     

Gene transfer into intact vertebrate embryos.

Intact chick embryos at 40 h incubation were transfected in vivo with chimeric vectors expressing chloramphenicol acetyl transferase (CAT) under different promoter sequences. The cationic lipid, dioctadecylamidoglycyl spermine (DOGS) used as the transfecting agent had no noticeable toxic effects on embryonic development. CAT activity was monitored 48 h post-transfection on homogenates of embryos dissected free of all annexes. Of the various constructs tested, those containing the AP-1 response element linked to CAT (TRE-tk-CAT) gave high expression and consistent enzyme responses within groups. Co-transfection experiments in which embryos were exposed simultaneously to a CAT vector containing the cAMP response element and to a vector expressing the catalytic subunit of protein kinase A showed that the promoters of the introduced genes can be regulated by their respective transacting factors. This method may therefore represent a general tool for introducing genes into intact vertebrate embryos at precise developmental times.     

Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors

Transposable elements can be considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion. The plasmid-based transposon system of Sleeping Beauty (SB) combines the advantages of viruses and naked DNA molecules. In contrast to plasmid vectors, transposons integrate through a precise, recombinase-mediated mechanism into chromosomes, providing long-term expression of the gene of interest in cells. The advantages of transposons in comparison to viral systems include their simplicity and improved safety/toxicity profiles. In addition, the hyperactive SB100X is the first plasmid-based delivery system that overcomes the efficacy of non-viral delivery. The transposon delivery system consists of the transposase and the integration cassette, recognized by the transposase. The plasmid-based transposon delivery system can be combined with any non-viral delivery method. Here we provide two detailed protocols to apply SB-mediated, non-viral gene transfer in cultured cells. In our first example, we use a lipid-based delivery method in combination with the transposon-based integration system in an easy-to-transfect (HeLa) cell line. Second, we show how to achieve 40–50% stable expression of a transgene in clinically relevant, hard-to-transfect cells (hematopoetic stem cells, HSCs) by nucleofection. The given protocols are adaptable to any vertebrate cells in culture.     

Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates

Receptor-associated protein (RAP) is a ligand for all members of low-density lipoprotein (LDL) receptor families. RAP is internalized into cells via receptor-mediated endocytic trafficking, making it an attractive mechanism for efficient gene delivery. In this study, we have developed a gene delivery system using RAP as a targeting ligand. A RAP cDNA lacking a C-terminal heparin-binding domain was amplified by polymerase chain reaction (PCR) from a human liver cDNA library and was reamplified by using a primer containing a cysteine codon at its carboxyl end to facilitate its conjugation to polylysine (polyK). RAP was purified using a bacterial expression system and coupled to poly-D-lysine (PDL) or poly-L-lysine (PLL) of average MW 50 kDa via the heterobifunctional cross-linker SPDP. Using fluorescence-labeled RAP ligand, cellular uptake of the transfection complexes into HepG2 cells was shown to be highly efficient and more specific to PDL-conjugated RAP compared with PLL-conjugated one. Plasmid DNA containing a luciferase reporter gene was condensed with either RAP-PDL or RAP-PLL. In vitro transfection into HepG2 cells with RAP-PDL conjugate resulted in significantly higher luciferase expression levels in comparison to either nonconjugated PDL, or RAP-PLL, or LipofecAMINE/DNA complexes in the presence of 10% fetal bovine serum. Luciferase expression was inhibited by the addition of excess RAP. Treatment of the cells with Lovastatin, which inhibits HMG-Co reductase and increases expression of LDL receptor, stimulates luciferase expression, suggesting that the gene delivery is specifically mediated by LDL receptor. Thus, RAP-PDL conjugates have the potential to be used as a new nonviral gene delivery vector.