Lentiviral Expression, Product Brochure

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Lentiviral Expression, Product Brochure

Interest in any of the products,

request or order them at Bio-Connect.

Lentiviral Expression Procedure

Purify your Lentivirus (p. 6)

Infect Target Cells (p. 7)

Measure Viral Titer (p. 4-5)

Package Virus with Gene of Interest (p. 2-3)

Lentiviral ExpressionLentiviral Expression

Choose your Viral Vector for Gene Delivery Adenovirus Adeno-

Associated Virus (AAV)

Lentivirus (HIV-1, FIV,

SIV)

Retrovirus (MMLV)

Gene Expression Transient Transient or

Stable Transient or Stable

Stable

Infect Dividing Cells Yes Yes Yes Yes

Infect Non-dividing Cells Yes Yes Yes No

Integration into Target Cell Genome No Yes Yes Yes

Relative Viral Titer XXXX XXX XXX XX

Relative Transduction Efficiency XXXX XXX XXX XX

Immune Response in Target Cells High Very Low Low Moderate

LENTIVIRAL EXPRESSION

ViraSafe™ Lentiviral Expression and Packaging Systems

Safely generate high-titer lentivirus with advanced 3rd-generation expression system

2 www.cellbiolabs.com

Lentivirus Production using the ViraSafe™ Lentiviral Expression System.

Lentivirus based on HIV-1 is a powerful means of gene transfer because of its ability to infect both dividing and non-dividing cells. Recently devel-oped third-generation lentiviral expression sys-tems have reduced the risk of creating replication-competent virus upon recombination, but the risk is still present. Our ViraSafe™ Lentiviral Expression Systems pro-vide a safer method even compared to other third-generation lentivirus systems. Sequence homology has been reduced substantially, thereby substan-tially reducing the risk of replication-competent len-tivirus. Each vector is provided separately to allow for optimization of vector ratios.

Safer to Use: 80-90% less sequence homol-ogy than other 3rd-generation lentiviral ex-pression systems; ecotropic systems available for even more safety*

High Titers: Incorporates elements that pro-vide titers comparable to other 3rd-generation systems

Economical: Vectors provided separately, not as a mixture, to allow optimization of vector ratios

More Flexible: Order only the components you need: Expression Vectors Packaging Vectors Complete Expression Systems (most

economical)

*Ecotropic lentiviruses may only readily infect mouse and rat cells, and will not readily infect humans or other species

When packaging your lentivirus, consider our 293LTV Lentiviral Cell Line. This cell line is de-rived from the parental HEK 293 cell line, but has been specifically selected for use in lentiviral expression. You’ll see firmer attachment to culture plates, flattened morphology, and a much lar-ger surface area for higher viral yields and superior transfection.


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ViraSafe™ Lentiviral Expression Systems

ViraSafe™ Lentiviral Packaging Systems

ViraSafe™ Lentiviral Expression Vectors

Cloning / Expression Vector

X X

Envelope Vector X X

Gag-Pol Vector X X

pRev Vector X X

LacZ Control Vector X

Comparison of ViraSafe™ Lentiviral Systems

Application

Best value: all the plasmids you need to make a recom-binant virus containing your

gene of interest

Co-transfect these plasmids with any 3rd generation lentiviral expression vector

Clone your gene of interest here and co-transfect with any 2nd or 3rd generation lentiviral packaging system

Selecting your Lentiviral Plasmids

First determine which lentiviral plasmids you need to construct your virus, then use the table below.

When selecting a ViraSafe™ Expression System or Packaging System, you have the option of an eco-tropic or pantropic system: Recombinant lentiviruses made using a ViraSafe™ Ecotropic System will only readily infect mouse

and rat cells; if you plan to use your lentivirus for only these species, you may feel safer knowing the lentivirus you make will not readily infect human cells.

Recombinant lentiviruses made using a ViraSafe™ Pantropic System are pseudotyped with VSVG as the envelope protein; such viruses can universally transduce cells of any species, but the probability of creating a replication-competent lentivirus is still reduced compared to viruses made with other systems.

When selecting a ViraSafe™ Expression System or Expression Vector, you can choose from 10 different expression vectors:

If you need a recombinant lentivirus with a reporter gene, choose one of our Premade Reporter Lentivirus Controls. These premade lentiviruses have a variety of uses including troubleshoot-ing any issues with infectivity of your target cells. Reporter Lentivirus Controls contain GFP, RFP, or ß-Galactosidase.

Promoterless vector miRNA vector with GFP/Puromycin fusion Dual promoter with puromycin Dual promoter with neomycin Dual promoter with hygromycin

Bicistronic (IRES) with GFP Bicistronic (IRES) with blasticidin Bicistronic (IRES) with puromycin Bicistronic (IRES) with neomycin Bicistronic (IRES) with hygromycin

Additionally, we offer a variety of reporter plasmids suitable for construction of recombinant reporter lenti-viruses. A complete listing of our ViraSafe™ vectors and systems may be found on pages 8-9.



Once you’ve harvested your lentiviral supernatant, you will want to measure the titer of your virus prior to infecting your target cells. This is espe-cially important when working with lentivirus be-cause of its tendency toward lower infection effi-ciencies. We offer three simple methods to quan-tify your lentivirus, each of which provides certain benefits.

QuickTiter™ Lentivirus Quantitation and Titer Kits

Eliminate bias from your lentivirus quantitation with easy-to-follow protocols

Most published method: Traditional p24 ELISA

The most published method for lentivirus quantita-tion involves measuring the lentiviral p24 nucleo-capsid protein. Our QuickTiter™ Lentivirus Quanti-tation Kit (p24 ELISA) provides a convenient method to measure the titer of your HIV-1 or FIV-based lentivirus in a standard microplate reader.

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Culture medium GFP LentiviralSupernatant

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p24 Titer of GFP Lentiviral Supernatant using the Quick-Titer™ Lentivirus Quantitation Kit (HIV p24 ELISA).

More accurate method: Lentivirus-Associated p24 ELISA

Though the traditional p24 ELISA is a well-published lentivirus quantitation method, it does have its flaws. Most lentiviral supernatants contain at least some free p24 protein not associated with the virus; in some cases the free p24 can com-prise up to 90% of the total p24 in the supernatant. Obviously this can bias your results and lead to an overestimation of your lentiviral titer. Our QuickTiter™ Lentivirus Titer Kit is an advanced version of the p24 ELISA. This kit uses an exclu-sive proprietary technology that efficiently sepa-rates free p24 from lentivirus-associated p24 in your supernatant. You can then apply your lenti-virus supernatant to the p24 ELISA, confident in the fact that your data will accurately reflect only p24 protein associated with your lentivirus.

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p24 (ng/mL)

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p24 in supernatant

p24 in pellet

Free p24 in Lentiviral Supernatants does not Complex with ViraBind™ Pulldown Reagents in the QuickTiter™ Lenti-virus Titer Kit (Lentivirus-Associated p24).

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QuickTiter™ Lentivirus Titer Kit

(Virus-Associated p24)

QuickTiter™ Lentivirus Quantitation

Kit (HIV or FIV p24 ELISA)

QuickTiter™ Lentivirus

Quantitation Kit

Assay Principle p24 ELISA with proprietary technology to separate free

p24 from viral p24 p24 ELISA

Measures nucleic acid content

Key Benefit Accuracy Most Published Speed (45-60 min.)

Viruses Detected HIV-1 HIV-1, FIV HIV-1, FIV, SIV

Detection Method Colorimetric (ELISA)

plate reader Colorimetric (ELISA)

plate reader Fluorescence plate reader

Kit Size 96 assays 96 assays 20 assays

Select your QuickTiter™ Quantitation Kit

Fastest method: Nucleic acid content

If speed is what you’re after, the QuickTiter™ Lenti-virus Quantitation Kit (#VPK-112) is made for you. This kit measures the viral nucleic acid content in your lenti-virus supernatant. In just under 1 hour, you can measure the physical titer of your virus on a fluo-rescence plate reader.

Assay Principle for the QuickTiter™ Lentivirus Quantitation Kit.

Assay Principle for the QuickTiter™ Lentivirus Titer Kit (Lentivirus-Associated p24).



ViraBind™ Lentivirus Concentration and Purification Kits

Concentrate and purify your lentivirus supernatant quickly and easily for superior infection efficiency

Purification of your lentivirus is important in obtain-ing good gene expression results. However, the thought of tedious, time-consuming centrifugation might tempt you to skip this crucial step. Filter-based kits are faster, but can only process small volumes of supernatant at a time. ViraBind™ Lentivirus Concentration and Purifica-tion Kits make purification fast, easy and highly efficient. These kits deliver high-yield lentivirus in as little as 6 hours. Obtain highly pure lentivirus with >60% recovery rate. Concentrate your virus up to 500-fold to 109 - 1010 TU/mL, which is suffi-cient for in vivo studies. ViraBind™ Purification Kits are available with two purification technologies: Gel filtration column followed by concentrator Dialysis apparatus followed by a concentrator;

this technology requires fewer steps and less hands-on time than the column format

ViraBind™ Lentivirus

Concentration and Purification Kit

ViraBind™ PLUS Lentivirus

Concentration and Purification Kit

ViraBind™ PLUS Lentivirus

Concentration and Purification Mega Kit

Method Column + Concentrator Dialysis + Concentrator Dialysis + Concentrator

Total Time 6-8 hours 10-24 hours 10-24 hours

Capacity per Prep (Lentiviral Supernatant)

100 mL 50 mL 500 mL

Kit Sizes 2 Preps 5 Preps

25 Preps 2 Preps

2 Preps 10 Preps

Select your ViraBind™ Purification Kit

ViraBind™ PLUS Lentivirus Concentration and Purifi-cation Kit Procedure.

Tip: After purifying your virus, it is a good practice to measure your viral titer again,

regardless of the purification method used.

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After you’ve determined the titer of your purified lentivirus, it is time to infect your target cells. The efficiency of transduction with lentivirus may be low in many target cells, leading to poor levels of gene expression. Adding chemicals such as Polybrene® can help somewhat, but may not be enough for low-abundance genes or cells that are more difficult to infect. The ViraDuctin™ Lentivirus Transduction Kit over-comes this obstacle to infection. A proprietary re-agent cocktail forms a supercomplex with the lenti-virus; the complex is pelleted away from the su-pernatant and is therefore removed from inhibitors that may reduce infection efficiency. The result is up to 6-fold higher transduction efficiency depend-ing on the target cell line.

ViraDuctin™ Lentivirus Transduction Kit

Easily increase lentiviral infection efficiency in your target cells

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NIH3T3 HeLa 293 HT-1080

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rmal

ized

GF

P F

luo

resc

ence

Polybrene

ViraDuctin™

Comparison of Relative Lentiviral Transduction Efficiencies of Various Cell Lines using Polybrene or the ViraDuctin™ Lentivirus Reagent Cocktail.

Comparison of GFP Lentiviral Transduction Efficiencies of Two Cell Lines using No Enhancer, Polybrene, or the ViraDuctin™ Lentivirus Reagent Cocktail.



Ordering Information and Published Citations

Lentiviral Expression Systems

Product Name Envelope Size / Qty Catalog Number

ViraSafe™ Universal Lentiviral Expression System (Promoterless) Ecotropic 1 Kit VPK-211-ECO

Pantropic (VSVG) 1 Kit VPK-211-PAN

ViraSafe™ Lentiviral Expression System (Puro) Ecotropic 1 Kit VPK-212-ECO


ViraSafe™ Lentiviral Expression System (Neo) Ecotropic 1 Kit VPK-213-ECO


ViraSafe™ Lentiviral Expression System (Hygro) Ecotropic 1 Kit VPK-214-ECO


ViraSafe™ Bicistronic Lentiviral Expression System (Puro) Ecotropic 1 Kit VPK-215-ECO


ViraSafe™ Bicistronic Lentiviral Expression System (Neo) Ecotropic 1 Kit VPK-216-ECO


ViraSafe™ Bicistronic Lentiviral Expression System (Hygro) Ecotropic 1 Kit VPK-217-ECO


ViraSafe™ Bicistronic Lentiviral Expression System (GFP) Ecotropic 1 Kit VPK-218-ECO


ViraSafe™ Bicistronic Lentiviral Expression System (Blasticidin) Ecotropic 1 Kit VPK-219-ECO


Ecotropic 1 Kit VPK-220-ECO

Pantropic (VSVG) 1 Kit VPK-220-PAN ViraSafe™ miRNA Lentiviral Expression System

Each of these complete third generation expression systems contains 5 individual plasmids: 3 packaging vectors (Gag-Pol, Rev, and Envelope), an expres-sion vector, and a LacZ control vector.

Lentiviral Expression Vectors

Product Name Size / Qty Catalog Number

pSMPUW Universal Lentiviral Expression Vector (Promoterless) 10 µg VPK-211

pSMPUW-Puro Lentiviral Expression Vector 10 µg VPK-212

pSMPUW-Neo Lentiviral Expression Vector 10 µg VPK-213

pSMPUW-Hygro Lentiviral Expression Vector 10 µg VPK-214

pSMPUW-IRES-Puro Lentiviral Expression Vector 10 µg VPK-215

pSMPUW-IRES-Neo Lentiviral Expression Vector 10 µg VPK-216

pSMPUW-IRES-Hygro Lentiviral Expression Vector 10 µg VPK-217

pSMPUW-IRES-GFP Lentiviral Expression Vector 10 µg VPK-218

pSMPUW-IRES-Bsd Lentiviral Expression Vector 10 µg VPK-219

pSMPUW miRNA Lentiviral Expression Vector 10 µg VPK-220

Each of these expression vectors may be used in conjunction with any 2nd or 3rd generation lentiviral packaging system.

Recent Product Citation Sankaran, V. et al. (2011). MicroRNA-15a and 16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13. PNAS 10.1073/pnas.1018384108. (VPK-212)

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Lentiviral Packaging Systems Each of these third generation packaging systems contains 3 individual plasmids: Gag-Pol, Rev, and Envelope (ecotropic or VSVG).

Product Name Envelope Size / Qty Catalog Number

ViraSafe™ Lentiviral Packaging System Ecotropic 1 Kit VPK-205

Pantropic (VSVG) 1 Kit VPK-206

Recent Product Citation Shimamura, T. et al (2008). Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance. Cancer Res. 68:5827-5838. (LTV-100)

Lentivirus Cell Line Product Name Size / Qty Catalog Number

293LTV Cell Line 106 cells LTV-100

Lentiviral Reporter Plasmids Product Name Size / Qty Catalog Number

pLenti-GFP Lentiviral Control Vector 10 µg LTV-400

pLenti-RFP/Puro Lentiviral Control Vector 100 µL LTV-403

pSMPUW-GFP/Puro Lentiviral Control Vector 10 µg LTV-401

pSMPUW-MNDnLacZ Lentiviral Control Vector 10 µg LTV-402

Lentivirus Plasmids for iPS Cell Reprogramming Product Name Size / Qty Catalog Number

pLentG-KOSM Lentiviral Vector 100 µL LTV-700

pLenti-p53 (Human) shRNA Lentiviral Vector 100 µL LTV-451

Premade Reporter Lentivirus Controls Product Name Concentration Size / Qty Catalog Number

GFP Lentivirus Control 1 x 106 TU/mL 200 µL LTV-300

RFP Lentivirus Control 1 x 106 TU/mL 200 µL LTV-301

ß-Galactosidase Lentivirus Control 1 x 105 TU/mL 500 µL LTV-302



Recent Product Citations 1. Sanchez-Antequera, Y. et al. (2011). Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target

cells. Blood 117:E171-E181. (VPK-107) 2. Tiedemann, R.E. et al. (2010). Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid

restricted kinase, GRK6. Blood 115:1594-1604. (VPK-107) 3. Keck, Z.-Y. et al. (2009). Mutations in HCV E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but

compromise virus infectivity. J. Virol. 83:6149-6160. (VPK-107) 4. Sukumaran, S. et al. (2009). Functional characterization of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-

phosphate acyltransferase isoform 3. J. Mol. Endocrinol. 42:469-478. (VPK-107) 5. Joshi, M.B. et al. (2009). Extracellular cadherin repeat domains EC1 and EC5 of T-cadherin are essential for its ability to stimulate angiogenic

behavior of endothelial cells. FASEB J. 23:4011-4021. (VPK-107) 6. Lin, R.-J. et al. (2009). Distinct antiviral roles for human 2’5’-oligoadenylate synthetase family members against dengue virus infection. J. Immu-

nol. 183:8035-8043. (VPK-107) 7. Dyer, K. et al. (2008). Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J. Immunol.

181:4004-4009. (VPK-107) 8. Escamilla-Hernandez, R. et al. (2008). Constitutively active protein kinase A qualitatively mimics the effects of follicle-stimulating hormone on

granulosa cell differentiation. Mol. Endocrin. 22(8):1842-1852. (VPK-107) 9. Iftikhar, M. et al. (2011). Lysyl oxidase-like-2 (LOXL2) is a major isoform in chondrocytes and is critically required for differentiation. J. Biol.

Chem. 286:909-918. (VPK-108-HIV-P24) 10. Agrawal-Gamse, C. et al. (2010). Yeast-elicited cross-reactive antibodies to HIV Env glycans efficiently neutralize virions expressing exclusively

high-mannoseN-linked glycan.s J. Virol. 10.1128/jvi.01349-10. (VPK-108-HIV-P24) 11. Piedrahita, D. et al. (2010). Silencing of CDK5 reduces neurofibrillary tangles in transgenic Alzheimer’s mice. J. Neurosci. 30:13966-13976. (VPK

-108-HIV-P24) 12. Young, A.P. and A.J. Wagers (2010). Pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of

canonical myogenic regulatory factors. J. Cell Sci. 123:2632-2639. (VPK-108-HIV-P24) 13. Kaletsky, R. et al. (2009). Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein. PNAS 106:2886-2891.

(VPK-108-HIV-P24) 14. Krishnamarchary, B. et al. (2009). Noninvasive detection of lentiviral-mediated choline kinase targeting in a human breast cancer xenograft.

Cancer Res. 69:3464-3471. (VPK-108-HIV-P24) 15. Malur, A. et al. (2009). Deletion of PPAR-gamma in alveolar macrophages is associated with Th-1 pulmonary inflammatory response. J. Immu-

nol. 182:5816-5822. (VPK-108-HIV-P24) 16. Shu, L. and P.J. Houghton (2009). The mTORC2 complex regulates terminal differentiation of C2C12 myoblasts. Mol. Cell Biol. 29:4691-4700.

(VPK-108-HIV-P24) 17. Agrawal-Gamse, C. et al. (2009). Adaptive mutations in a human immunodeficiency virus type 1 envelope protein with a truncated V3 loop re-

store function by improving interactions with CD4. J. Virol. 83:11005-11015. (VPK-108-HIV-P24) 18. Lambert, M.P. et al. (2009). Platelet factor 4 regulates megakaryopoiesis through low density lipoprotein receptor related protein 1 (LRP1) on

megakaryocytes. Blood 114:2290-2298. (VPK-108-HIV-P24) 19. Brittain, J.M. et al. (2009). An atypical role for collapsing response mediator protein (CRMP-2) in neurotransmitter release via interaction with

presynaptic voltage-gated calcium channels. J. Biol. Chem. 284:31375-31390. (VPK-108-HIV-P24) 20. Wilson, A.A. et al. (2008). Sustained expression of -1 antitrypsin after transplantation of manipulated hematopoietic stem cells. Am. J. Respir.

Cell Mol. Biol. 39:133-141. (VPK-108-HIV-P24) 21. Apolonia, L. et al. (2007). Stable gene transfer to muscle using non-integrating lentiviral vectors. Mol. Ther. 15:1947-1954. (VPK-108-HIV-P24) 22. Sadat, S. et al. (2007). The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF. Biochem. and Biophys. Res.

Comm. 363:674-679. (VPK-108-HIV-P24) 23. Thomassen, M.J. et al. (2007). ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar

proteinosis. J. Lipid Res. 48:2762-2768. (VPK-108-HIV-P24) 24. Veeraraghavalu, K. et al. (2010). Presinilin 1 mutants impair the self-renewal and differentiation of adult murine subventricular zone-neuronal

progenitors via cell-autonomous mechanisms involving notch signaling. J. Neurosci. 30:6903-6915. (VPK-112) 25. Niwano, K. et al. (2008). Lentiviral vector-mediated SERCA2 gene transfer protects against heart failure and left ventricular remodeling after

myocardial infarction in rats. Mol. Ther. 16:1026-1032. (VPK-112)

Lentivirus Quantitation & Titer Kits Product Name Detection Size / Qty Catalog Number

QuickTiter™ Lentivirus Titer Kit (Lentivirus-Associated p24) Colorimetric 96 Assays VPK-107

5 x 96 Assays VPK-107-5

QuickTiter™ Lentivirus Quantitation Kit (FIV p24 ELISA) Colorimetric 96 Assays VPK-108-FIV-P24

5 x 96 Assays VPK-108-FIV-P24-5

QuickTiter™ Lentivirus Quantitation Kit (HIV p24 ELISA) 96 Assays VPK-108-HIV-P24

5 x 96 Assays VPK-108-HIV-P24-5

QuickTiter™ Lentivirus Quantitation Kit Fluorometric 20 Assays VPK-112

Colorimetric


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Lentivirus Purification / Concentration Kits Product Name Capacity/Prep Size / Qty Catalog Number

ViraBind™ Lentivirus Concentration and Purification Kit 100 mL

2 Preps VPK-090

5 Preps VPK-091

25 Prep VPK-091-5

ViraBind™ PLUS Lentivirus Concentration and Purification Kit 50 mL 2 Preps VPK-095

ViraBind™ PLUS Lentivirus Concentration and Purification Mega Kit 500 mL 2 Preps VPK-096

10 Preps VPK-096-5


Lentivirus Transduction Kits Product Name Size / Qty* Catalog Number

40 Transductions LTV-200

200 Transductions LTV-201 ViraDuctin™ Lentivirus Transduction Kit

*Based on use in a 24-well plate. The kit may also be used with 96-well, 12-well or 6-well plates, as well as 60mm or 100mm dishes. See product insert for possible number of transductions for each format.

Recent Product Citations 1. McEachron, T.A. et al. (2010). Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis. Blood 116:5037-5044. 2. Zemskova, M. et al. (2010). P53-dependent induction of prostate cancer cell senescence by the PIM1 protein kinase. Mol. Cancer Res. 8:1126-

1141.


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Tel: 1-858-271-6500 Toll-Free: 1-888-CBL-0505 Fax: 1-858-271-6514

Trademark Information QuickTiter, ViraBind, ViraDuctin and ViraSafe are trademarks of Cell Biolabs, Inc. Polybrene is a registered trademark of Abbott Laboratories.

Lentiviral Expression, Product Brochure

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