mirPS® Liposomal Kit User Manual
Contents
1. Sox2 and Nanog but also have a highly demethylated genome similar to hES cells Microarray analyses revealed that genome wide gene expression patterns between the mirPS and hES H1 and H9 cells share over 86 92 similarity Under molecular guidance in vitro these mirPS cells can differentiate into many distinct tissue cell types derived from all three germ layers Therefore miR 302 can replace Oct4 Sox2 and Nanog for simpler and more efficient generation of iPS cell lines support mellobiotech com TEL 562 946 0131 FAX 562 645 5332 Liposomal Kit For delivery of miR 302 only or miR 302 367 into mammalian cells Included in this kit Additional Equipment and Reagents Required miR 302 or miR 302 367 Plasmid l Mammalian cells with appropriate culture medium Control Plasmid Collagenase trypsin EDTA solution 4 1 optional Transfection Reagent Cell culture incubator at 37 C under 5 CO Tissue culture dishes Feeder Free Culture Medium M 800 01 1 1 mg mL puromycin stock PROTOCOL Step 1 Cell Preparation 1 Seed appropriate number of mammalian cells onto a 100 mm tissue culture dish The number of cells required varies with cell type For best results wait until cells have adhered to the plate s surface and are about 40 60 confluent on the day of transfection 2 One hour prior to transfection replace the cell culture medium with 10 ml of fresh serum free antibiotic free medium Step 2 Transfecti2. usage of this kit is subject to the US and PCT patent laws 3 This kit is to not be used for human cloning or generation of hybrids mirPS mir 302 Transfection Kits and mirPS mir 302 367 Transfection Kits mirPS Kits are covered under a series of international patents and patents pending Rights to use these products are limited to research only They are not to be used for any other purpose including but not limited to use in drugs use in diagnostic use in therapeutics or in use for human cloning or generation of hybrids This limitation expressly excludes the right to resell or otherwise transfer the mirPS Kits or its component parts to third parties These products may not be modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval in advance from Mello Biotech Notwithstanding the above academic and not for profit research institutions whose research using the mirPS Kits is sponsored by for profit organizations which shall receive ownership to data and results stemming from the sponsored research shall need a separate commercial license agreement from Mello Biotech in order to use the mirPS Kits Inquiry into the availability of a license for commercial or therapeutic purposes please contact a licensing representative by phone at 562 946 0131 or by e mail at license mellobiotech com mirPS is a registered trademark of Mello Biotechnology Inc Mello Biotech
3. References Lin et al 2008 MiR 302 reprograms human skin cancer cells into a pluripotent ES cell like state RNA 14 2115 2124 2 Lin et al 2010 MicroRNA miR 302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4 6 cell cycle pathways Cancer Res 70 9473 9482 _ 3 Lin et al 2011 Regulation of somatic cell reprogramming through inducible mir 302 expression Nucleic Acids Res 39 1054 1065 4 Moyoshi et al 2011 Reprogramming of mouse and human cells to pluripotent using mature microRNAs Cell Stem Cell Vol 8 633 638 5 Anokye Danso et al 2011 Highly efficient miRNA mediated reprogramming of mouse and human somatic cells to pluripotency Cell Stem Cell 8 376 388 6 Subramanyam et al 2011 Multiple targets of miR 302 and miR 372 promote reprogramming of human fibroblasts to induced pluripotent stem cells Nat Biotech 29 443 448 7 Card et al 2008 Oct4 Sox2 regulated miR 302 targets cyclin D1 in human embryonic stem cells Mol Cell Biol 28 6426 6438 Notice to Purchaser CAREFULLY READ THE FOLLOWING TERMS AND CONDITIONS BEFORE OPENING THIS KIT OPENING THIS PACKAGE INDICATES YOUR ACCEPTANCE OF THESE TERMS AND CONDITIONS IF YOU DO NOT AGREE WITH THEM YOU SHOULD PROMPTLY RETURN THE PACKAGE UNOPENED AND YOUR MONEY WILL BE REFUNDED 1 Upon using this kit the users agree to the terms of the Notice to Purchaser and government regulations 2 All
4. cells with 50 100 ug mL puromycin for 24 48 hours Observe cells if a large percentage of non transfected cells remain increase the puromycin concentration and treat for and additional 24 48 hours or until population is relatively pure 2 Once iPSC colonies are larger select colonies either manually with a pipette or with a micromanipulator and transfer them to a new tissue culture dish containing Feeder Free Culture Medium Mello Bio cat no M 800 011 Allow colonies to grow to desired size Troubleshooting Does the miR 302 367 plasmid integrate into the genome Integration depends on the delivery methods Liposomal transfection is transient in nature and it should not integrate into the genome Transfection by electroporation should not cause integration However under conditions of high voltage or long duration some integration lt 10 may occur Lentiviral infection leads to integration D 125 302 PR For laboratory research use ONLY Not for therapy or diagnostics Not for Resale How can I improve the transfection efficiency GFP should be observed within 1 2 days after transfection Some cells are difficult to transfect and may require multiple transfections Low transfection efficiency may be the result of multiple factors including e Inappropriate cell density at the time of transfection e Inadequate incubation time for the transfection complex e Poor cell condition old cells If you are experiencing problems with lipos
5. logo and trademarks are the property of Mello Biotechnology Inc Copyright 2009 by Mello Biotechnology Inc D 125 302 PR For laboratory research use ONLY Not for therapy or diagnostics Not for Resale mirPS Liposomal Kit User Manual Catalog Numbers M 125 302 CM M 126 302 CM M 127 302 CM M 128 302 CM Stable for six months when stored at 4 C Introduction cPPT Pew miR 302 or miR 302 367 a pUC ori Figure 1 The miR 302 Liposomal Expression Kit is designed for intracellular delivery and expression of the miR 302 microRNA family miR 302b c a and d pre microRNA cluster with or without miR 367 As shown in Figure 1 expression of the miR 302 pre microRNA is cytomegalo virus CMV promoter driven and directly linked to GFP expression allowing easy identification of transfected cells In addition the plasmid includes a puromycin resistance gene for selection of target cells The miR 302 family is expressed most abundantly in early human embryonic stem hES cells and quickly decreases after cells differentiate Transgenic delivery of miR 302s into human skin hair follicle and kidney epithelial cells as well as prostate cancer breast cancer liver cancer and melanoma cells has been shown to orchestrate the reprogramming of somatic cells to induced pluripotent stem iPS like cells References 1 2 and 3 The miR 302 reprogrammed iPS cells namely mirPS cells not only express many key hES cell markers such as Oct4
6. omal transfection try increasing or decreasing the amount of transfection complex used per reaction Mello Biotech also offers an electroporation kit which may work better with some cell lines What is the morphology of a miR 302 induced pluripotent stem cell The iPSCs are spherical cells with loose nuclei and are surrounded by abundant cytoplasm I have initial high transfection efficiency but very few iPSC colonies How can increase the reprogramming efficiency Reprogramming is largely dependent on cell type and concentration of miR 302 Lin et al NAR 2011 Make sure puromycin selection is performed Wait 3 4 days after Puromycin selection before transferring colonies to Feeder Free Medium If you switch cells to Feeder Free Medium too soon cells may detach and form aggregates which may reduce iPSC survival We recommend culturing iPSC colonies under feeder containing conditions to improve reprogramming efficiency How can isolate an iPSC colony to produce a cell line A two to eight cell stage iPSC colony is the best choice to isolate for the formation of a single iPSC line Cell isolation can be performed under an inverted microscope using a micromanipulator in conjunction with a micro injector holder Alternatively you can select colonies with pipette and very sharp eyes Fluorescence activated cell sorting FACS can also be used to isolate iPSC colonies How do I ensure the quality of miR 302 plasmid Store the kit at 80 C free
7. on 1 Into a sterile 1 5 mL microcentrifuge tube add 15 uL of miR 302 Plasmid or Control Plasmid 2 Add1 mL of serum free antibiotic free medium and mix well by vortexing Spin tube briefly 3 Add 50 uL of Transfection Reagent 3 to the center of the 1 5 mL microtube Add directly into the liquid in the tube Do not pipette the transfection reagent onto the sides of the tube 4 Vortex for at least 30 seconds For best results when preparing more than one transfection complex at the same time be sure to mix each tube immediately after adding the transfection reagent 5 Incubate for 10 15 minutes at room temperature The transfection mixture can be incubated for longer at room temperature but do not exceed 30 minutes 6 Add the mixture drop wise covering the entire surface of the 100 mm culture dish Mix by shaking the culture dish back and forth and right to left several times to evenly distribute the transfection mixture 7 Incubate the cells at 37 C under 5 COs for 12 18 hours and then replace the medium with FBS containing growth medium 8 Continue to grow the cells at 37 C under 5 COs until the green positive cells can be observed under a fluorescent microscope GFP expression is typically observed1 2 days post transfection The results may vary with different cell types Step 3 Selection of Positive Cells 1 Once GFP expression has been observed puromycin selection may be used to remove any non transfected cells Treat
8. zer to maintain its quality The plasmid is stable for one year under this condition Avoid repeated freeze thaw cycles of the plasmid Due to the structural complexity of the miR 302 cluster amplification of the plasmid may cause mutations If you would like to sequence the plasmid to insure its quality please contact Mello Biotech for primer information Refer to the FAQ section of our website http www mellobiotech com faq html and Lin et al 2011 Nucleic Acids Research 39 1054 1065 for additional information on miR 302 367 reprogramming support mellobiotech com TEL 562 946 0131 FAX 562 645 5332
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