Inducible Reprogramming Lentiviral Vectors Expressing
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1. 5 GFP cells gated 0 0001 0 0010 0 0100 0 1000 0 2 0 4 dilution dilution Notice to purchaser Purchaser represents and warrants that it will use the Vectalys Constitutive Reprogramming Lentiviral Vectors Human OSKM purely for research purposes not for diagnostic use not for resale and not for use in humans or veterinary applications Vectalys will not be held responsible for patent infringement or other violations that may occur with the use of our products Purchaser must determine the suitability of the product s for their particular use Additional terms and conditions may apply COMPANY Vectalys SAS Canal Biotech II 3 rue des Satellites 31400 Toulouse S 33 5 61 28 70 75 fax 33 5 62 26 12 44 E vectalys vectalys com2. your gene transfer partner functional and nonfunctional particles Calculating the volume of vectors from this type of titration may distort the results The key factor normalization of viral vector titers using a reporter expressing vector as an internal standard The viral titer determined by Vectalys corresponds to the number of integrated genomes qPCR or transduction units FACS generated by transducing 1E5 HCT116 cells with 1ml of viral supernatant in our experimental conditions 3 days of cell culture after transduction with 8ug ml of Polybrene The results obtained by qPCR critically depend on the conditions of the titration experiment It is therefore crucial to include standard controls with the samples to standardize the titers from one quantification experiment to another This normalization method using an internal qPCR control in all titrations experiments allows the titers of vector batches to be comparable with each other The internal control corresponds to viral vectors expressing reporters The titer obtained from FACS experiments corresponds to the level of expression of the reporter proteins and may be considered as a functional titer This result is used to correlate viral titers obtained by qPCR biological titer with viral titer obtained by FACS functional titer It is then possible to deduce the functional titer from the viral titer obtained by qPCR for any samples QPCR R 0 9901 w 5 e YN a o O
3. NI Product Specification Sheet 20131203 ve ct a IVs your gene transfer partner rectal ys your gene transfer partner Inducible Reprogramming Lentiviral Vectors Expressing Polycistronic Human OSKM for Somatic Cell Reprogramming www vectalys com Products COMPANY Vectalys SAS Canal Biotech II 3 rue des Satellites 31400 Toulouse S 33 5 61 28 70 75 fax 33 5 62 26 12 44 E vectalys vectalys com Product Specification Sheet 20131203 ve ct a IVs your gene transfer partner Inducible Reprogramming Lentiviral Vectors Catalog Number referring to this User Manual 0027VCT 0028VCT Contents Cat 0027VCT 3x20pl of rLV EF1a Tet On 3G IRES Neo WPRE lentiviral vectors Cat 0028VCT 3x20pl of rLV TRE3G OSKM WPRE inducible polycistronic lentiviral vectors Please refer to the certificate of Analysis CoA for the titer of your particular lot Product Description The Vectalys Inducible Reprogramming Lentiviral Vectors are prepackaged lentiviral particles expressing polycistronic Oct4 Sox2 KLF4 and Myc for reprogramming human somatic cells under the induction of doxycycline using Tet technology These VSV G pseudotyped viruses are capable of infecting both dividing and non dividing cells The high purification level of these lentiviral particles allows transduction of primary cells and immortalized cell lines without inducing any toxicity to the cells even at
4. amount of polycistronic TRE3G OSKM lentiviral vectors according to the thawing protocol provided above Homogenize the viral supernatant by pipeting up and down and not by inverting the vial upside down D Prepare the transduction mix by adding the required volume of thawed viral particles to complete media containing 20ul of Polybrene 800ug ml The final volume should be 4ml at maximum for transduction in a 6 well plate E Discard the medium from each well and add the transduction mix to the cells be careful to apply the transduction mix to the well edges to avoid any cell disruption Gently rock the plate from side to side to mix the viral vectors onto the target cells F Incubate the plate for 5h in a 37 C 5 CO incubator G After the 5 hour incubation discard the transduction mix and wash the cells once with 3ml 1x PBS per well Then discard the PBS and replace it with 4ml of complete media H Four days post transduction perform a second transduction using Tet 0n3G lentiviral vectors by repeating the steps described above A second transduction must not be performed before 48h after the first transduction I Once the two transduction performed seed the cells by plating from 1x10 to 5x10 cells per well of a 6 well plate according to the cell type you are using Use the same culture medium that is used to maintain target cells in a proliferative state Incubate overnight in a 37 C 5 CO incubator The next day add 50ng
5. high multiplicity of infection M 0 I The expression of these four factors has been shown to reprogram a variety of differentiated cells into induced pluripotent stem iPS cells with embryonic stem ES cell like properties Handling and Storage Store at 80 C Keep frozen until use Avoid repeated freezing and thawing The use of gloves and disposable lab coats while working with viral derived vectors is strongly recommended This product must only be handled in a biosafety cabinet under BSL 2 conditions Lentiviral vectors are stable for at least 1 year after receipt when stored at 80 C After thawing immediately place on ice please refer to the thawing protocol included in this document The viral vectors are packaged in working aliquots and can be thawed just before use In the case that more than one freeze thaw cycle is required according to your application Vectalys recommends to expect a decrease of about 15 20 in viral vector titer for each freeze thaw cycles This product is distributed for research use only It is not for use in diagnostic procedures as the safety and efficacy of this product in diagnostic or other clinical uses has not been established The use of lentiviral derived vectors requires you to follow laboratory biosafety procedures and practices in agreement with your country regulations SYMBOL CLASSIFICATION A Biorisk class C2L2 Safety precautions COMPANY Vectalys SAS Canal Biotech I
6. l 3 rue des Satellites 31400 Toulouse T 33 5 61 28 70 75 fax 33 5 62 26 12 44 E vectalys vectalys com Product Specification Sheet 20131203 ve ct a IVs your gene transfer partner The greatest safety risk associated with viral delivery systems comes from the potential generation of recombinant viruses that are capable of autonomous replication during the packaging process The Vectalys Reprogramming Lentivirus platform eliminates these hazards by combining a disabled viral genome with a unique manufacturing process Also the viral genes that facilitate the enclosing of the sequence in a viral capsid e g Gag Pol Env are distributed on multiple helper plasmids which do not contain significant regions of homology during packaging This strategy further minimizes the probability of recombination events that might otherwise generate viruses capable of autonomous replication With these safety measures the Vectalys Reprogramming Lentivirus particles can be used in standard Biosafety Level 2 tissue culture facilities and should be treated with the same level of caution as any other potentially infectious agent Also please note that c Myc and KIf4 proteins are reported to have oncogenic properties Directions for use thawing protocol The vectors should be taken out of the 80 C freezer and placed on ice immediately prior to use Thaw the vectors on ice Once thawed the vectors should be used f
7. ml of doxycycline to induce OSKM expression At each medium change add 50ng ml of doxycycline until the appearance of iPS clones COMPANY Vectalys SAS Canal Biotech II 3 rue des Satellites 31400 Toulouse S 33 5 61 28 70 75 fax 33 5 62 26 12 44 E vectalys vectalys com gt Product Specification Sheet 20131203 ve Cta IVs your gene transfer partner Quality controls The several different quality controls are applied to the Vectalys Reprogramming Lentiviral Vectors The viral vectors are tittered in efficient transduction units Viral integration copy numbers are quantified by transduction of mammalian cells followed by qPCR of the genomic DNA The viral vectors are also tittered as physical particles using an ELISA assay The specific activities according to DNA and proteins impurities are measured The residual DNA and total proteins are quantified using commercial kits These controls are performed because Vectalys has demonstrated the impact of residual DNA and protein impurities on increasing cellular toxicity after transduction Frequently Asked Questions What is the difference between transduction units TU and physical particles PP During the production of viral vectors functional and nonfunctional particles are produced A functional and complete particle is called a transduction unit TU Physical Particles PP represents functional particles and als
8. o empty particles damaged particles and free p24 protein see figure below The PP TU ratio is a quality control measure of the quality of the vector product It is optimal to remove as much PP in the viral supernatants to keep only the functional particles It is therefore necessary to have reliable and discriminating titration methods for the different types of particles PP Efficient Inefficient transducing particules Free capsid transducing proteins particle Why use a titer expressed in TU ml A titer in TU ml is required for accurate calculation of functional vectors applied to the cells The TU ml titer provided by Vectalys is a precise measure of only functional viral vectors Calculating the volume of vectors from this type of titration is precise and the experimental results can be extrapolated and compared from one experiment to another Number of cells seeded Viral vectors volume required ul _______ X M O l x 1000 Viral Vectors Titer TU ml Titration of physical particles PP is often used in commercial kits by competitors because its ease of use But the PP ml titer provides an approximate titer of viral vectors because it represents a mixture of COMPANY Vectalys SAS Canal Biotech II 3 rue des Satellites 31400 Toulouse 33 5 61 28 70 75 fax 33 5 62 26 12 44 E vectalys vectalys com gt Product Specification Sheet 20131203 ve Cta IVs
9. or transduction as soon as possible to avoid degradation 1 Just before transduction remove the tubes of viral supernatant from the 80 C freezer and thaw them on ice at 4 C 2 It is essential to avoid thermal shock to the cells and vectors If the vectors will be diluted in medium use medium that has been equilibrated to room temperature to minimize the heat shock to the vectors and the cells 3 Five minutes before transduction remove the tubes from ice and allow to warm to room temperature In the case that more than one freeze thaw cycle is required according to your application expect a decrease of about 15 20 in viral vector titer for each freeze thaw cycle Directions for use Materials Required but Not Provided 6 well plates TC grade Cell counter hemocytometer Complete media FBS supplemented Phosphate Buffered Saline PBS Polybrene Hexadimethrine bromide Sigma 107689 10G UN Ph WwW N Directions for use Transduction Protocol for Human Somatic Cells The following protocol has been optimized for early passage human foreskin fibroblasts This protocol allowed us to transduce human foreskin fibroblast cells with a transduction efficiency of 100 Use the following protocol as a reference to optimize conditions that will enable the generation of iPS cells from other human target cells The pre transduction seeding density and cell quantity to be transduced can be modified as necessary The multiplicit
10. y of infection M 0 I the time of viral vector incubation on cells and the Polybrene concentration are critical points that must strictly be followed COMPANY Vectalys SAS Canal Biotech II 3 rue des Satellites 31400 Toulouse S 33 5 61 28 70 75 fax 33 5 62 26 12 44 E vectalys vectalys com Product Specification Sheet 20131203 ve ct a IVs your gene transfer partner Day 0 Cells seeding Seed the cells by plating from 1x10 to 5x10 cells per well of a 6 well plate according to the cell type you are using Use the same culture medium that is used to maintain target cells in a proliferative state The maximum volume per well of a 6 well plate should be 4ml Incubate overnight in a 37 C 5 CO incubator Be sure to include a control well for counting the number of cells on the day of transduction Day1 Transduction A Count the number of cells in the control well of the 6 well plate The control well is used to determine the amount of viral vectors needed to achieve the target M 0 I and to keep the M 0 I constant from one experiment to another B Using the following equation determine the volume of each viral vector required to achieve the advised M O I of 5 Please pay attention to the lot titer as it may vary by lot Number of cells seeded Viral vectors volume required ul _ X M O I x 1000 Viral Vectors Titer TU ml C Thaw the required
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