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amaxa eNews #2
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Nucleofection of Somatic Stem Cells
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Stem cells have the remarkable potential to develop into many different cell types in the body, and as they can theoretically divide without limit, somatic stem cells are generally believed to serve an essential role in replenishing and repairing damaged tissues. These characteristics make stem cells the ideal cell type for research into future therapies for a wide range of disorders as well as general understanding of cell fate determination and differentiation pathways.
Nucleofection is a highly efficient non-viral alternative to transiently transfect somatic stem cells and so is the ideal tool to assist you in your stem cell research, whether it be in studying differentiation patterns or characterizing the basic metabolic processes of these cells.
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Your experiments will benefit from:
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| Non-viral technology | No biosafety concerns
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| High efficiencies | Over 70% transfection efficiency with high cell viabilities
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| Substrate flexibility | Identical transfection conditions for transfection of DNA, mRNA, siRNA, shRNA vectors, miRNA etc.
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| Optimized protocols | Individually optimized Nucleofector Kits available for the most popular cell types
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| Versatility | Nucleofector Basic Kits allow nucleofection of cells for which optimized protocols do not yet exist
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| Functionality | Unaltered cell differentiation potential after nucleofection demonstrated for many cell types
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Mouse embryonic stem (ES) sells
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The ability of embryonic stem cells to generate complete animals or to be differentiated into any of the individual tissues makes them applicable for an immense range of research fields. Nucleofection helps to make the most of these options by providing an increased uptake and expression of transfected DNA without compromising the ability of the cells to create chimeric or 100% ES-derived mice [Application note: Boljahn et al.].
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Somatic stem cells
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While ES cells maintain the potential to differentiate into any cell type, directing them to differentiate down specific lineages can be quite complicated. Because of this, specific precursor cells are often more appropriate for many research applications. amaxa offers specific protocols and kits for the nucleofection of:
Human Mesenchymal Stem Cells (MSC) - pluripotent progenitor cells obtained from bone marrow stroma that posess the ability to generate cartilage, bone, muscle, tendon, ligament and fat.
Nucleofection is able to provide transfection efficiencies as high as 70% in these cells without interfering with their ability to differentiate into cells such as adipocytes and osteoblasts [Aluigi et al. (2006) Stem Cells 24(2):454-461].
Human CD34+ cells - derived from blood or bone marrow, this population includes both hematopoetic stem cells and lineage-committed progenitor cells, making them the ideal vehicle for the study of a wide range of blood and immune disorders.
Nucleofection efficiencies of up to 70% can be obtained in these cells, equalling levels otherwise obtainable only by using retroviral methods [Papetrou et al. (2005) Gene Therapy 12:118-130], without influencing hematopoetic cell differentiation [Wiehe et al. (2006) Regenerative Med. 1(2): 223-234].
Mouse and rat Neural Stem Cells (NSC) - isolated from neural tissue, these cells can be differentiated into a variety of neural and glial cell types. The potential ability to generate new neural tissue has raised considerable interest by researchers studying a wide variety of neurological disorders with the hope of developing a means to eventually replace damaged or diseased neural tissue.
Nucleofection provides the first non-viral means to reproducibly achieve high transfection efficiencies in neural stem cells without compromising their functionality or ability to be subsequently differentiated into different cell types [Copray et al. (2006) Stem Cells 24(4):1001-1010 and application note].
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Figure: Average transfection efficiencies for somatic stem cells achieved with nucleofection. Data show average transfection efficiencies of human mesenchymal stem cells (MSC), human CD34+ progenitor cells, rat and mouse neural stem cells (NSC), nucleofected with either H-2Kk or GFP expressing plasmids. After 20-60 hours transfection efficiency was determined by flow cytometry.
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Basic Nucleofector Kits expand the range of cells which can be nucleofected
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Although optimal nucleofection results for most primary cells require very particular conditions, amaxa has recently developed a series of Basic Nucleofector Kits in order to help you find suitable conditions for your cells of interest.
Customers have used these kits successfully for human and bovine neural stem cells (Basic Neuron Nucleofector Kit) and others.
Other progenitor cell types which have been successfully nucleofected include:
Mouse Muscle-Derived Stem Cells (MDSC) - Quenneville et al. (2004) Molecular Therapy, 10(4): 679-687
Human Myoblasts - Quenneville et al. (2004) Molecular Therapy, 10(4): 679-687
Rat Neural Stem-Like Cells (NSLC) - Kondo and Raff (2004) Genes Dev 18(23): 2963-2972
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Ordering information
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| Cat.No. | Devices | | AAD-1001 | Nucleofector II Device | | AAM-1001 | 96-well Shuttle
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| Cat.No. | Nucleofector Kits for specific primary cell types | | VPA-1003 | Human CD34+ Nucleofector Kit | | VPH-1001 | Mouse ES Cell Nucleofector Kit | | VPE-1001 | Human MSC Nucleofector Kit | | VPG-1004 | Mouse NSC Nucleofector Kit | | VPG-1005 | Rat NSC Nucleofector Kit | | | | | Cat.No. | Basic Nucleofector Kits for primary cells | | VPI-1001 | Basic Endothelial Cell Nucleofector Kit | | VPI-1002 | Basic Fibroblast Cell Nucleofector Kit | | VPI-1003 | Basic Neuron Nucleofector Kit | | VPI-1004 | Basic Smooth Muscle Cell Nucleofector Kit | | VPI-1005 | Basic Epithelial Cell Nucleofector Kit |
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Differentiation of nucleofected rat NSC
Primary rat neural stem cells isolated from rat embryos (E14) were nucleofected using the Rat NSC Nucleofector Kit, program A-31 and a plasmid encoding the enhanced green fluorescent protein eGFP under control of an EF1a promoter. After nucleofection, the cells were cultured with bFGF for 2 days, then for 5 days without bFGF to differentiate NSC into neurons and astrocytes. Cells were analyzed by fluorescence microcopy 2 days (A) and 7 days post nucleofection (B). (Photograph courtesy of Dr. S. H. Lee, College of Medicine, Dept. of Biochemistry, Hanyang University, Seoul, South Korea).
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amaxa's NucleofectionŽ Process, NucleofectorŽ Device and NucleofectorŽ Solutions are covered by PCT Applications PCT/EP01/07348, PCT/DE02/01489, PCT/DE02/01483, and other pending patents, and domestic or foreign applications corresponding thereto.
amaxa, Nucleofector, Nucleofection, maxGFP and 96-well Shuttle are trademarks of amaxa AG. Other product and company names mentioned herein are the trademarks of their respective owners.
amaxa disclaims all warranties, whether expressed or implied, including any warranty as to the quality, accuracy, safety, or suitability of the information provided in this e-newsletter for any particular purpose. Any use of the information contained on any page of this e-newsletter is evidence of agreement with these terms of use.
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