Premade Lentiviral Particles for human ips Stem Factors

Transcription

1 Premade Lentiviral Particles for human ips Stem Factors For generating induced pluripotent stem (ips) cells or other applications RESEARCH USE ONLY, not for diagnostics or therapeutics Cat# Product Name LVP003 h OCT4 (RFP-Bsd) inducible particles LVP004 h SOX2 (RFP-Bsd) inducible particles LVP005 h NANOG (RFP-Bsd) inducible particles LVP006 h LIN28 (RFP-Bsd) inducible particles LVP007 h Myc (RFP-Bsd) inducible particles LVP008 h Klf4 (RFP-Bsd) inducible particles LVP-stems-h-RB Full set (6 human stem factors-with RFP-Bsd marker) h OCT4 (Neo) inducible particles LVP311 LVP312 LVP313 LVP314 LVP315 LVP316 LVP-stems-h-N LVP317 LVP318 LVP319 h SOX2 (Neo) inducible particles h NANOG (Neo) inducible particles h LIN28 (Neo) inducible particles h cmyc (Neo) inducible particles h KLF4 (Neo) inducible particles Full set (6 human stem factors-with Neomycin marker) h OCT4 (EF1α) (puro) particles h SOX2 (EF1α) (puro) particles h NANOG (EF1α) (puro) particles amounts 200ul/ea x 6 200ul/ea x 6

2 LVP320 LVP321 LVP322 LVP588 LVP589 LVP590 LVP591 LVP592 LVP593 LVP-stems-h-EF1apuro LVP-stems-h-EF1a- RP h LIN28 (EF1α) (puro) particles h Myc (EF1α (puro) particles h Klf4 (EF1α) (puro) particles Full set (6 human stem factors driven by EF1α promoter with puromycin marker) h OCT4 (EF1α) (RFP-Puro) particles h SOX2 (EF1α) (RFP-Puro) particles h NANOG (EF1α) (RFP-Puro) particles h LIN28 (EF1α) (RFP-Puro) particles h Myc (EF1α (RFP-Puro) particles h Klf4 (EF1α) (RFP-Puro) particles Full set (6 human stem factors driven by EF1α promoter with RFP-Puromycin marker) 200ul/ea x 6 200ul/ea x 6 Storage: < -70 C, avoid repeat freeze/thaw cycles. Products stable for 6 month. Product Description: Lentiviral system is a gene delivery tool using lentivectors for gene expression or knockdown. Lentivectors are HIV-1 (Human Immunodeficiency Virus 1) derived plasmids, used to generate lentiviral particles (lentivirus) that can be transduced into virtually all mammalian cell types or organs, including stem cells, primary cells and non-dividing cells both in vivo and in cell culture system. Particles stably integrate into the transduced cell s genome for long term expression. Therefore, lentivirus holds unique promise as a gene transfer agent. Converting fully differentiated mouse or human somatic cells into embryonic-like cells (so called induced Pluripotent Stem Cell: ipsc) has attracted enormous attention in stem cell research. Multiple reports have demonstrated that ips cells were generated by using a set of transcription factors or stem cell factors that are delivered as expression virus or expressed proteins. Although the combination of reprogramming factors may slightly different, the main stem cell factors are: OCT3/4, SOX2, NANOG, LIN28, cmyc and KLF4.

3 Amsbio provides two sets of premade lentivirial particles for human or mouse ips genes. Each stem factor is natively expressed (without any tags) under either an optional inducible sucmv promoter (set#1) or enhanced EF1a promoter (set#2). Six human stem cell factors were first individually cloned into lentivector. Then, lentivectors were cotransfected with a packaging mix (Cat# HT-pack) into a 293T packaging cells (cat# TLV-C). The premade lentiviral particles are VSV-G pseudotyped virus, packaged in DMEM medium, and supplied as 200ul/per vial at ~ 1x 10 7 IFU/ml. All six stem factor have been verified by sequencing. Their sequences fully match to the CD region according to the NCBI s database (see table below). Target NCBI ID Matched ORF position h Myc NM_ h Klf4 NM_ h Oct3/4 NM_ h SOX2 NM_ h LIN28 NM_ h NANOG NM_ The set #1 has a tetracycline inducible sucmv promoter to drive ips gene expression. It also contains a selection marker (RFP-Bsd fusion dual marker or Neomycin marker) under RSV promoter (see vector map scheme below). These particles can be used for regular constitutive high expression. And the same particles can also optionally used as tetracycline induced expression when the tetracycline regulator protein (TetR) is present in advance. For inducible expression, the TetR must be expressed in advance to stop the transcription, and the expression is activated by adding tetracycline. This inducible expression is tetracycline s dose dependent. In general, the amount of tetracycline used is 1ug/ml final concentration. Please see the schematic instruction (below) for the mechanism of

4 inducible expression. Amsbio provides premade lentivirus expressing TetR with different antibiotic markers. sucmv Tet operator gene Expression particles tetr protein tet Expression repressed Add tet ( ) Expression induced 1. tetr protein binds to tetoperator sequences in promoter to repress the transcription. 2. Tetracycline binds to tetr to release tetr from promoter, and transcription is initiated. How tetracycline induction works 2. The set #2 has the enhanced constitutive EF1α promoter to drive ips gene expression (see vector map scheme below) with the options to use either a RFP-Puromycin fusion dual marker or puromycin marker.

5 Safety Precaution: Amsbio lentiviral particles have adopted the most advanced lentiviral safety features (using the third generation vectors with self-inactivation SIN-3UTR), and the premade lentivirus is replication incompetent. However, please use extra caution when using lentiviral particles. Use the lentiviral particles in Bio-safety II cabinet. Wear gloves at all times when handling Lentiviral particles! Please refer CDC and NIH s guidelines for more details regarding to safety issues. Related Products: Pre-made lentivirus Products: Product Category 0TUFluorescent protein U0T 0TUHuman and mouse ORFsU0T 0TULuciferase expressionu0t 0TUCRE recombinaseu0t 0TULoxP ColorSwitchU0T 0TUTetR inducible expression Product Description (please Category name to see product's pages) Premade Lentivirus for GFP/ CFP/ YFP/ RFP Premade lentivirus expressing human and mouse ORFs with RFP-Blasticidin fusion dual marker. Premade lentivirus for luciferase protein expression: firefly and Renilla with different antibiotic selection markers. Premade lentivirus for expression of nuclear permeant CRE recombinase with different flurescent and antibiotic markers. Premade lentivirus expressing "LoxP-GFP-Stop-LoxP-RFP" cassette, used to monitor the CRE recombination event in vivo. Premade lentivirus expressing TetR (tetracycline regulator) protein, the repressor protein for the inducible expression system. repressoru0t T-antigen Expression 0TUCell Organelle imagingu0t Expression of large and small T antigen with different selection markers Premade lentivirus for cell organelle imaging. The fluorescent marker GFP/RFP/CFP is localized in different cell organelle for living cell imaging.

6 LacZ expression 0TUFluorescent-ORF fusionu0t 0TUPre-made shrna lentivirusu0t 0TUmicroRNA and anti-microrna lentivirusu0t 0TUNegative control lentivirusesu0t Other Enzyme expression Expression of full length β- galactosidase (lacz) with different selection markers Pre-made lentivirus expressing a "GFP/RFP/CFP-ORF" fusion target. Premade shrna lentivirus for knockdown of specific genes (P53, LacZ, Luciferase and more). Premade lentivirus for of expression human or mouse precursor mirna. And antimirna lentivector and virus for human and mouse mirna. Premade negative control lentivirus with different markers: serves as negative control for lentivirus treatment, for validation of the specificity of any lentivirus target expression effects. Ready-to-use lentivirus, expressing a specific enzyme with different selection markers. References: 1. Masaki Ieda, Ji-Dong Fu, et al. (2010). Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell 142, Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, Park, I.H., et al., Reprogramming of human somatic cells to pluripotency with defined factors. Nature, (7175): p Shao, L., et al., Generation of ips cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame. Cell Res., (3): p NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors link 7. CDC guidelines for Lab Biosafety levels link

7 Attachment: ips cell generation procedure (Dox inducible system) (as reference only) Day 0: Seed the parent cells: For example, seed human fibroblast cells at cells/well in 6-well plate, cultured in 5ml of growth medium, incubated overnight at 37 C with 5% CO 2. Day 1: Viral Transduction: On the second day, remove medium, add 2.5 ml of pre-warmed fibroblast growth medium, then add 500ul of ips lentivirus. Gently mix for evenly distribution, then incubate overnight at 37 C with 5% CO 2. [Note: set up inducible GFP positive control well by adding 200ul/per well of GFP control particles.] Day 2: Change Medium: At about 24 hours post-transduction, change medium with 5 ml of growth medium. Incubate overnight at 37 C with 5% CO 2. Day 3: Re-plate the transduced cells to feeder cells After three days trypsinize the cells, centrifuge at 200 x g for 5 minutes, resuspend in Fibroblast Cell Growth Medium, and re-plate in a 150mm MEF Feeder Dish. Incubate cells overnight at 37 C with 5% CO 2. Day 4: Induce Reprogramming using Doxycycline: At 24 hours after re-seeding, fibroblast Cell Growth Medium is replaced with 2.0 ml of Dox-Induction Medium containing 2ug/ml of Dox. Cells are incubated overnight at 37 C with 5% CO 2. [Note: set up negative control well without Dox.] Day 5+: Change induction Medium: Change of Dox-Induction Medium every 48 hours, continue to pass the cells until they showed typical human ES cell morphology. Day 14++: Select ips cell colonies: According to proper cell morphology, pick the ips cell colonies using a sterile glass picking tool. Trypsinize each individually isolated ips cell colony and pass into each well in a 24-well feeder plate. Passage, and Expand ips cell colonies: Incubate 24-well plate at 37 C and 5% CO 2, replace with fresh culture medium without Dox every 48 hours. Also passage into an appropriate size for ips cell expansion (the process takes about 6-10 days). Monitor ips cell colony growth and morphology, and validate the ips colonies. Save ips cell in cryogenic vials.

8 ips cell sample images: