GeneCopoeia HPK-LvTR-20, HPK-LvTR-40 User Guide
G Cn po eiaee o
TM
Expressway to Discovery
G Cn po eiaee o
TM
Expressway to Discovery
Lenti-Pac™ HIV Expression Packaging Kit
For optimized production of recombinant lentivirus
Cat. No. HPK-LvTR-20 (20 transfections)
Cat. No. HPK-LvTR-40 (40 transfections)
User Manual Version V
GeneCopoeia, Inc.
9620 Medical Center Drive, #101
Rockville, MD 20850
USA
301-762-0888 or 866-360-9531
inquiry@genecopoeia.com
www.genecopoeia.com
© 2009 GeneCopoeia, Inc.
Lenti-Pac™ HIV Expression Packaging Kit User Manual
USER MANUAL
Lenti-Pac™ HIV Expression Packaging Kit
I. Introduction
II. Kit Contents and Storage
III. Additional Materials Required or Recommended
IV. Getting Started
V. Lentivirus Production
VI. Lentivirus Titer Estimation by Transduction
VII. Transduction of Target Cells with Lentiviruses
VIII. Limited Use License and Warranty
I. Introduction
GeneCopoeia has multiple sets of over 40,000 human and mouse ORF expression clones as well as multiple sets
of small hairpin RNAi (shRNA) clones against genome-wide target genes from human, mouse, rat, and other
mammals in HIV-based lentiviral vector systems. In addition, GeneCopoeia offers precursor microRNA (miRNA)
expression and miRNA inhibitor clones for all known human, mouse and rat miRNAs in a HIV-based lentiviral vector
system. HIV- (human immunodeficiency virus) based vectors are currently the most popular lentiviral-based
expression systems and are very effective at transducing genes into a wide variety of dividing and non-dividing
mammalian cells, both in vitro and in vivo. The lentiviral expression vectors can integrate into the genome of the
target cells, resulting in the stable expression of transgenes. The GeneCopoeia third generation HIV-based
lentivector systems meet Biosafety Level 2 (BSL-2) requirements based on the criteria published by the Centers for
Disease Control.
The GeneCopoeia Lenti-Pac™ HIV Expression Packaging System includes an optimized lentiviral packaging
plasmid mix, an eGFP positive control plasmid, a new transfection reagent, EndoFectin™ optimized for virus
production and TiterBoost™ reagent that further increases the titers 5-10 fold. When combined with GeneCopoeia
HIV-based lentiviral constructs the results are high titers and robust expression levels. The Lenti-Pac HIV
Expression Packaging System safely ensures efficient expression of recombinant transcripts in mammalian cells.
Advantages of OmicsLink™ lentiviral ORF expression and shRNA clones, miExpress™
precursor miRNA expression and miArrest™ miRNA inhibitor clones
High efficiency of gene delivery to virtually all mammalian cell types in vitro as well in vivo
High expression levels of delivered genes (ORF expression clones)
High knockdown efficiency against target mRNA transcripts (shRNA clones)
High expression translation suppression of target genes and/or mRNA cleavage/degradation (miRNA clones)
Self-inactivation and no unwanted viral replication
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Lenti-Pac™ HIV Expression Packaging Kit User Manual
Contents
Quantity
Shipping
temperature
Storage temperature/ conditions
HIV packaging mix
100 μl (0.5 μg/μl)
200 μl (0.5 μg/μl)
Ambient
4–8°C (Stable for at least 6 months)
Alternatively, the packaging mix can be
stored at -80 °C in usable aliquots.
Avoid repeated freezing/ thawing.
eGFP positive
control plasmid
25 μl (0.06 μg/μl)
25 μl (0.06 μg/μl)
Ambient
4–8°C (Stable for at least 6 months)
Alternatively, the packaging mix can be
stored at -80 °C in usable aliquots.
Avoid repeated freezing/ thawing.
EndoFectin Lenti
transfection reagent
500 μl
1ml
Ambient
4–8°C (Stable for at least 12 months)
TiterBoost™
Reagent (500x)
500 μl
1ml
Ambient
4–8°C (Stable for at least 12 months)
II. Contents and Shipping/ Storage
Contents and storage recommendations for the Lenti-Pac HIV Expression Packaging Kits
(Cat. No. HPK-LvTR-20/HPK-LvTR-40) are provided in the following table.
III. Additional Materials Required or Recommended
1. GeneCopoeia GCI-L3 chemically competent cells (GeneCopoeia Cat No. STK300-10). Alternatively, Stbl3™
chemically competent cells (Invitrogen Cat No. C7373-03) may be used.
2. GeneCopoeia 293Ta Lentiviral packaging cell line (GeneCopoeia Cat No. Clv-PK-01). Alternatively, the
HEK 293T/17 cell line (ATCC Cat No. CRL-11268) can also be used.
3. H1299 cell line (ATCC Cat No. CRL-5803), or HT-1080 cell line (ATCC Cat No. CCL-121) for lentivirus titer
estimation. H1299 cells are preferred over HT-1080 cells for lentivirus titration.
4. DMEM with glucose, L-glutamine and sodium pyruvate (Mediatech Cat No. 10-013-CV)
5. Fetal bovine serum (Thermo Scientific Cat No. SH300700.02)
6. Opti-MEM® I Reduced-Serum Medium (Invitrogen Cat No. 31985-062/31985-070).
7. Polybrene® (Sigma-Aldrich Cat No. H9268): 10 mg/ml solution dissolved in 150 mM NaCl and sterile-filtered.
Store usable aliquots at -20oC. Working stock can be stored at 4oC for up to two months.
8. Crystal Violet (Sigma-Aldrich Cat No. C3886): 0.5% (W/V) solution dissolved in 25% methanol.
9. Penicillin-Streptomycin for mammalian cell culture (Sigma-Aldrich Cat No. P4333)
10. Antibiotics for selecting stably transduced cells: puromycin (Invivogen Cat No. ant-pr-1/ant-pr-5), hygromycin
B (Invivogen Cat No. ant-hm-1/ant-hm-5), Neomycin (G-418) (Invivogen Cat No. ant-gn-1/ant-gn-5)
11. BD Falcon® 5-ml or 14-ml Tubes (BD Falcon Cat No. 352053/352059).
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