Gene transfer strategies for the physiologist
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Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3',5'-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organismKeywords:
Transgenesis
Transduction (biophysics)
Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14–15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14–15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.
Transgenesis
Blastoderm
Chorioallantoic membrane
Heterologous
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Abstract Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. We established a new method of eggshell windowing for embryo manipulation at different developmental stages. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.
Transgenesis
Blastoderm
Chorioallantoic membrane
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Here, we describe the production of transgenic quail via a germline transmission system using postmigratory gonadal primordial germ cells (gPGCs). gPGCs retrieved from the embryonic gonads of 5-day-old birds were transduced with a lentiviral vector and subsequently transferred into recipient embryos. Testcross and genetic analyses revealed that among three germline chimeric G0 quail, one male produced transgenic offspring; of 310 hatchlings from the transgenic germline chimera, 24 were identified as donor-derived offspring, and 6 were transgenic (6/310, 1.9%). Conventional transgenesis using stage X blastodermal embryos was also conducted, but the efficiency of transgenesis was similar between the two systems (<1.6 vs. 1.9% for the conventional and gPGC-mediated systems, respectively). However, substantial advantages can be gained from gPGC-mediated method in that it enables an induced germline modification, whereas direct retroviral transfer to stage X embryos causes mosaic integration. The use of gonadal PGCs for transgenesis may lead to the production of bioreactors.
Transgenesis
Chimera (genetics)
Germline mosaicism
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Gene delivery, a process of introducing foreign functional nucleic acids into target cells, has proven to be a very promising tool for inducing specific gene expression in host cells. Many different technologies have been developed for efficient gene delivery. Among them, electroporation has been adopted in gene delivery for decades, and it is currently widely used for transfection of different types of cells. Despite of the success achieved by bulk electroporation (BEP) for gene delivery in vitro and in vivo, it has significant drawbacks such as unstable transfection efficacy and low cell viability. In recent years, there is an emerging interest in understanding how individual cell accepts and responds to exogenous gene materials using single cell based micro-/nano-electroporation (MEP/NEP) technologies. In this review, the authors provide an overview of the recent development of MEP/NEP and their advantages in gene delivery. Additionally, the future perspectives of gene delivery with the application of electroporation are discussed. Keywords: Electroporation, gene delivery, nano-electroporation, nanocarriers, microfluidics.
Nanocarriers
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Transduction (biophysics)
Gene transfer
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To avoid safety issues such as immune response and cytotoxicity associated with viruses and liposomes, physical methods have been widely used for either in vivo or ex vivo gene delivery. They are, however, very invasive and often provide limited efficiency. Using pEGFP and pSEAP plasmids and NIH 3T3 fibroblasts as models, we demonstrate a new electroporation-based gene delivery method, called membrane sandwich electroporation (MSE). The MSE method is able to provide better gene confinement near the cell surface to facilitate gene transport into the cells and thus shows significant improvement over transgene expression of mammalian cells compared to current electroporation techniques.
Ex vivo
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Transgenesis
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