Efficient production of germline transgenic chickens using lentiviral vectors

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.     

Efficient production of transgenic chickens using self-inactive HIV-based lentiviral vectors

We demonstrated the simple and effective production of transgenic chickens,in which the enhanced green fluorescence protein(EGFP)was expressed by using third-generation self-inactive HIV-based lentiviral vectors.In our experiments,lentiviruses were injected into 204 fertilized eggs,from which 30(15%)chickens were hatched.The exogenous gene was detected in the genomes of 16 out of 30(53%)chickens.The green fluorescence signal was observed directly in various body parts,and was particularly significant in the...     

Efficient production of transgenic chickens using self-inactive HIV- based lentiviral vectors

We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein （EGFP） was expressed by using third-generation self-inactive HIV-based lentiviral vectors. In our experiments, lentiviruses were injected into 204 fertilized eggs, from which 30 （15%） chickens were hatched. The exogenous gene was detected in the genomes of 16 out of 30 （53%） chickens. The green fluorescence signal was observed directly in various body parts, and was particularly significant in the testes. The transgenes were also found in the offspring of these chickens. The results indicate that HIV-based lentiviral vectors can be used to generate transgenic birds economically and effectively [ Current Zoology 55 （5）： 383 - 387,2009].     

Ubiquitous GFP expression in transgenic chickens using a lentiviral vector

We report the first ubiquitous green fluorescent protein expression in chicks using a lentiviral vector approach, with eGFP under the control of the phosphoglycerol kinase promoter. Several demonstrations of germline transmission in chicks have been reported previously, using markers that produce tissue-specific, but not ubiquitous, expression. Using embryos sired by a heterozygous male, we demonstrate germline transmission in the embryonic tissue that expresses eGFP uniformly, and that can be used in tissue transplants and processed by in situ hybridization and immunocytochemistry. Transgenic tissue is identifiable by both fluorescence microscopy and immunolabeling, resulting in a permanent marker identifying transgenic cells following processing of the tissue. Stable integration of the transgene has allowed breeding of homozygous males and females that will be used to produce transgenic embryos in 100% of eggs laid upon reaching sexual maturity. These results demonstrate that a transgenic approach in the chick model system is viable and useful even though a relatively long generation time is required. The transgenic chick model will benefit studies on embryonic development, as well as providing the pharmaceutical industry with an economical bioreactor.     

Upscaling of lentiviral vector production by tangential flow filtration

BACKGROUND: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods. METHODS: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation. RESULTS: Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively. CONCLUSIONS: With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.     

Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector

Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.     

Prolonged liver-specific transgene expression by a non-primate lentiviral vector

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease.     

Efficient gene targeting mediated by a lentiviral vector-associated meganuclease

Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes.     

Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens

A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone (AS), different dilution concentrations of the bacterium and temperature during cocultivation were evaluated. A protocol was developed in which transient GUS expression activity was observed ranging from 25% to 55% out of the cocultivated FEC cultures, when FEC cultures were infected for 30 min with 50 μM AS, 1:10 dilution of bacteria, and then cocultivated at 24°C in the dark for 7 days with Agrobacterium strain LBA4404 (pTOK233) that carried gus, nptII and hpt genes. Seven independent experiments produced a total of 1300 transformed somatic embryos with shoots from 3.5 g of FEC. Of these germinated embryos, 50% developed into plants in vitro. Thus, on average, 500 mg of FEC infected with A. tumefaciens produced approximately 80–100 transgenic plants within 6–8 months via a selection process with 2.5–20 mg L^?1 hygromycin. Additionally, transformation was also performed with Agrobacterium strain AGL1 (containing the uidA and ppt genes), and this showed that luciferase‐based selection was less detrimental to the transgenic lines than was herbicide‐based selection. The transformation efficiency was 18.6% for the luciferase‐based selection and 7.6% for the PPT‐based selection, although with luciferase‐based selection, more false positives were obtained (about a quarter of the lines were escapes). The nptII and uidA genes were detected by polymerase chain reaction analysis in nine of the 19 tested lines. The results indicate that the system developed here can be used as an alternative to particle bombardment of Alstroemeria.     

Advances in transgenic rat production

Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.     

Enhanced lentiviral vector production in 293FT cells expressing Siglec-9

Siglecs, sialic acid-recognizing Ig-superfamily lectins, regulate various aspects of immune responses, and have also been shown to induce the endocytosis of binding materials such as anti-Siglec antibodies or sialic acid-harboring bacteria. In this study, we demonstrated that the expression of Siglec-9 enhanced the transfection efficiency of several cell lines such as macrophage RAW264 and non-hematopoietic 293FT cells. We applied this finding to the production of a lentiviral vector in which cells were transfected simultaneously with multiple vectors, and achieved a twice increase in viral production levels. Furthermore, 293FT cells expressing lectin-defective Siglec-9 produced three- to seven-fold higher titer of viral vector compared with parental 293FT cells. These results suggest that Siglec-9 enhanced lentiviral vector production in a lectin-independent manner.     

Efficient production of transgenic cattle by retroviral infection of early embryos

Production of transgenic cattle by microinjection of DNA has been difficult and costly. To explore an alternative method, one- to four-cell bovine embryos were exposed to a replication-defective retrovirus by microinjection of retrovirus producer cells into the perivitelline space. Embryos were cultured in vitro for 3–4 days, then transferred to recipient cows for further development. Thirteen of 22 embryos recovered at 15 days gestation and each of four fetuses recovered at 90 days gestation were transgenic. Fetuses harbored between 2 and 12 pro-viruses, and within each fetus, identical patterns of integration were observed in seven tissues tested. Estimates of the number of proviruses per cell suggested that in three of the four fetuses, most, and possibly all, cells were transgenic. This technique should facilitate application of transgenic technology to cattle and other agriculturally important species. © 1995 Wiley-Liss, Inc.     

Efficient production of transgenic chickens based on piggyBac

Transgenic techniques in chickens have been developed much more slowly than in mammals due to chickens’ unique reproduction mechanism. Retroviral methods have been the most successful. piggyBac (PB) is a transposon that has a 13 bp perfect terminal invert repeat sequence. PB can be inserted into TTAA sites and can also be precisely excised in mammals. Therefore, we have selected PB as a candidate to establish a new method to produce transgenic chickens. We constructed three donor vectors (ZGl-neo, ZGm-neo and ZGs-neo) expressing a GFP marker-gene and a neomycin resistant gene based on PB. We co-transfected each donor vector with a helper vector (CAG-PBase). We found that ZGl-neo was the most efficient PB vector. This vector could insert into TTAA sites in DF-1 cells. PB vectors were microinjected into sub-germinal cavity of newly laid eggs, and electroporation was then performed with a 20-V pulse for 5 cycles of 50 ms on and 100 ms off. GFP was expressed in different tissues of the embryos, including the gonads. Twenty-two chickens hatched after microinjection with compounds ZGl-neo and CAG-PBase (3:1). When we screened the blood DNA, 73 % (16/22) of the individuals were positive. Thirteen of the chickens grew to adulthood, 11 of which were males. 40 % (4/10) of the individuals were semen positive, and their copy numbers ranged from 0.05 to 0.21 (0.11, 0.21, 0.05, 0.06). No G1 offspring containing the integrated transposon were produced. We conclude that the PB transposon system is a novel useful tool for the efficient production of transgenic chickens.     

Efficient transgenesis in farm animals by lentiviral vectors

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.     

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII

Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.     

Efficient bacterial production of functional antibody fragments using a phagemid vector

The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.     

Correction of glycogenosis type 2 by muscle-specific lentiviral vector

Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid α-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. We investigated for the first time the use of lentiviral vectors to correct the GSDII phenotype in human and murine GAA-deficient cells. Fibroblasts from infantile and adult GSDII patients were efficiently transduced by a GAA-expressing lentiviral vector placed under the control of the strong MND promoter, leading to a complete restoration of enzymatic activity. We also developed a muscle-specific lentiviral vector based on the synthetic C5–12 promoter and tested it on deficient myogenic satellite cells derived from a GSDII mouse model. GAA was expressed as a correctly processed protein allowing a complete enzymatic and metabolic correction in myoblasts and differentiated myotubes, as well as a significant mannose-6-phosphate (M6P)-dependent secretion reuptake by naive cells. Transduced cells showed lysosomal glycogen clearance, as demonstrated by electron microscopy. These results form the basis for a therapeutic approach of GSDII using lentiviral vector-mediated gene transfer into muscle stem cells.