| Description | |
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| Catalogue Number | SCR526 |
| Description | Alzheimer’s In A Dish™ APPSL-GFP Lentivirus |
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| Background Information | Amyloid-beta plaques and neurofibrillary tangles are key pathological features observed in the brains of Alzheimer’s patients. Familial, early onset forms of AD (FAD) are caused by autosomal dominant inherited genetic mutations and offer an opportunity to study the effects of key mutations on the disease’s progression and pathology. To date, approximately 200 FAD mutations in APP and/or PSEN1 have been reported. Transgenic mouse models and human neurons harboring FAD mutations in PSEN-1 and/or APP are widely used as model systems and have provide important insights into the disease. Transgenic mouse models are able to recapitulate the loss in cognitive functions along with increased deposition of b-amyloid plaques, but are unable to demonstrate neurofibrillary tangles, one of the key pathological hallmarks of AD. Similarly human iPSC-derived neurons from FAD patients demonstrated increased levels of the pathogenic AB species and phosphorylated tau, but lack the characteristic amyloid-beta plaques and neurofibrillary tangles. Recently a 3D model using genetically engineered human neural stem cells that overexpress FAD mutations was reported to recapitulate the two pathological hallmarks of AD: b-amyloid plaques and neurofibrillary tangles. Lentiviruses expressing FAD mutations in human APP with both the K670N/M671L (Swedish) and V717I (London) mutations (APPSL) and/or PSEN1 with the E9 mutation (PSEN1(E9)) and APPSL/PSEN1(E9) along with fluorescent proteins as reporters for viral infection (see below), were used to transfect ReNcell VM human neural stem cells. FACS was utilized to enrich for cells with the highest transgene expressions followed by encapsulation of the sorted cells in a 3D Matrigel culture system. After approximately 6 weeks of differentiation, aggregation of AB was observed. Robust accumulation of phosphorylated tau along neurofibrillary tangles was readily detectable after 10-14 weeks. MilliporeSigma’s Alzheimer’s In A Dish™ APPSL-GFP Lentivirus is a necessary reagent to set up the Alzheimer’s 3D culture system. The lentivirus contains the APPSL FAD mutations and can be used to infect human neural cells. The TagGFP2 tag enables assessment of viral infectivity and allows FACS sorting of the highest expressing cells. References 1. Ryan NS, Rossor MN (2010) Correlating familial Alzheimer’s disease gene mutations with clinical phenotype. Biomark Med. 4(1): 99-112. 2. D’Avanzo C, Aronson J, Kim YH, Choi SH, Tanzi RE, Kim DY (2015) Alzheimer’s in 3D culture: challenges and perspectives. Bioassays 37(10): 1139-1148. 3. Choi SH, Kim YH, Hebisch M, Sliwinski C, Lee S, D’Avanzo C, Chen H, Hooli B, Asselin C, Muffat J, Klee JB, Zhang C, Wainger BJ, Peitz M, Kovacs DM, Woolf CJ, Wagner SL, Tanzi RE, Kim DY (2014) A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature 515(7526): 274-278. 4. Kim YH, Choi SH, D’Avanzo C, Hebisch M, Sliwinski C, Bylykbashi E, Washicosky KJ, Klee JB, Brüstle O, Tanzi RE, Kim DY (2015) A 3D human neural cell culture system for modeling Alzheimer’s disease. Nature Protocol 10(7): 985-1006. |
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| Components |
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| Quality Level | MQ100 |
| Applications | |
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| Application | The APPSL-GFP lentivirus can be used to generate 3D cell culture models of Alzheimer’s Disease. The high-titer polycistronic lentiviral particles contain the APPSL and TagGFP2 proteins and can be used to infect human neural stem cells at high efficiency. |
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| Biological Information | |
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| Product Usage Statements | |
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| Quality Assurance | The packaged lentivector constructs are provided as frozen VSV-G pseudotyped viral particles. The titer (ifus/ml) is defined by SBI’s Global UltraRapid Lentiviral Titer Kit (Cat. # LV961A-1) in HT1080 cells transduced by the packaged constructs. |
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| Storage and Shipping Information | |
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| Storage Conditions | Storage and Handling Lentivirus is stable for at least 6 months when stored at -80°C. After first thaw, place immediately on ice and store in working aliquots to avoid further freeze thaws. Avoid freeze thaws as this will result in a decrease in the virus titer. Important Safety Note: • Replication-defective lentiviral vectors are not known to cause any diseases in humans or animals. However, lentiviruses can integrate into the host cell genome and thus pose some risk of insertional mutagenesis. • Wear gloves when using this product. Avoid skin contact or ingestion of all reagents and chemicals used in this protocol. • Lentivirus is a risk group 2 and should be handled under approved Biosafety Level 2 (BSL2) controls. |
| Packaging Information | |
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| Material Size | 15ul/vial |