Dataset Information

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

ABSTRACT: Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was approximately 100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only approximately 1% with other gene delivery methods. Furthermore, in combination with negative selection, approximately 45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.

SUBMITTER: Suzuki K

PROVIDER: S-EPMC2544531 | biostudies-other | 2008 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

Suzuki Keiichiro K Mitsui Kaoru K Aizawa Emi E Hasegawa Kouichi K Kawase Eihachiro E Yamagishi Toshiyuki T Shimizu Yoshihiko Y Suemori Hirofumi H Nakatsuji Norio N Mitani Kohnosuke K

Proceedings of the National Academy of Sciences of the United States of America 20080903 37

Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cell ...[more]

PMID: 18768795

Similar Datasets

Project description:Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild-type adenovirus in those cells. Nevertheless, the incorporation of relatively low affinity peptide ligands into the fiber knob, while effective in vitro, has only minimal targeting efficacy in vivo and highlights the importance of high affinity ligand:receptor interactions to achieve tumor targeting.

Project description:Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes are greatly enhanced.

Dataset Information

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

Publications

Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets