Genetically Engineered Mice by Pronuclear DNA microinjection
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.
Current protocols in mouse biology
09/2012; 2:245-262. DOI: 10.1002/9780470942390.mo110168
The generation of transgenic mice by DNA microinjection is a powerful tool to investigate the molecular regulation of gene expression, development, and disease. The power of this technology is that foreign DNA can be introduced into every cell of a developing organism and the phenotypic impact of this genetic modification can be investigated in a system under the constraints of normal development and physiology. The generation of transgenic mice requires the preparation of the transgene DNA construction, collection of one-cell fertilized mouse embryos, injection of the transgene into mouse embryos, and transfer of the surviving embryos. Mice born from such manipulations are then screened for the presence of the transgene. The execution of these procedures requires a highly efficient system otherwise the cost of the generation of these mice can be cost prohibitive. However, the production of these animals can serve as an invaluable research resource.
Available from: Pan Tao
[Show abstract] [Hide abstract]
ABSTRACT: The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This "progene delivery" approach might lead to new types of vaccines and genetic therapies.
[Show abstract] [Hide abstract]
ABSTRACT: Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co-injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase-catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid-based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi-transposon transgenic mice in a single step.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.