1. What is a Lentivirus?Lentivirus is a subfamily of the retrovirus family. Lentiviruses can deliver significant amounts of genetic information into host cells and integrate it into the cellular genome. Genetically-engineered lentiviruses are therefore used as one of the most efficient tools of gene delivery.
These lentiviruses contain a viral promoter which is used to control the expression of a transgene or shRNA but no virulence genes anymore. Together with several other security modifications this makes them safe to use in the laboratory.
2. How does lentivirus work? Co-transfection of packaging plasmids and transfer vector into a packaging cell line allows efficient production of lentiviral particles which are released into the cells supernatant.
Viral particles harvested from the cell supernatant can transduce a wide range of both dividing and non-dividing mammalian cell types. Upon infection with lentiviral particles, the single stranded RNA (ssRNA) is reverse-transcribed and the resulting double-stranded DNA (dsDNA) stably integrates into the genome of the host providing for long term transcription of gene of interest or shRNA.
3. What kinds of premade lentiviral particle does Genemedi provide? Genemedi provides ready-to-use particles for shRNA expression and gene expression. Each particles contains a sequence fully verified shRNA or a specific gene target. Particles provided in either DMEM medium containing 10% FBS, or in PBS solution. Purified viruses in PBS are provided as in vivo ready status, suitable for in vivo applications, or for suspension cell transduction, and for transduction in cell lines requiring a serum-free culture conditions. Genemedi provides particles containing different antibiotic markers, including: Blasticidin (Bsd), puromycin (Puro), luciferase (Luc), neomycin (Neo), and fusion dual markers as: Bsd-GFP, Bsd-RFP, Puro- GFP, Puro-RFP and others. All Genemedi’s Lentiviral particles can be used for constitutive target (or shRNA) over-expression. Optionally, the same particles can be used as tetracycline inducible expression when a tetracycline regulator (tetR) protein is present (called optional inducible expression).
4. What's the optimal concentration of viruses that I should use for infection? It depends on the purpose of the experiment. Higher titer of lentivirus should be used for in vivo experiment compared to in vitro experiment. Though lentiviral vector is highly efficient in transduction, the transduction efficiency of lentivirus depends heavily on the cell type to be transduced. Pilot experiment is highly recommended to determine how efficient the lentivirus on your target cells.
5. How much culture media should I use during infection? For your reference, we recommend the following amount of virus-containing media for infection:
- 10-cm plate: 8-10 ml/plate
- 6-well plate: 1 ml/well
- 12-well plate: 0.5 ml/well
- 24-well plate: 0.2 ml/well
6. How do I determine the right amount of lentivirus to add to the target cells? One of the key factors of a successful transduction is the cell type. For example, transduction efficiency is much higher in actively dividing cells than in non-dividing cells. In addition, transduction of cells works better at lower MOI (multiplicity of infection) than at higher MOI. MOI is the ratio of the number of lentivirus particles to the number of cells. For some cell types, the higher the MOI , the larger the volume and higher the titer of lentivirus is required in order for the experiment to succeed. You can adjust the cell number and add the appropriate amount of lentivirus according to what has been reported in the scientific literature. If there is no adequate information in the scientific literature, we recommend performing a preliminary experiment using gradient dilutions of lentivirus, such as 0.1 μl, 0.3 μl, 0.5 μl, 0.7 μl, 0.9 μl for Genemedi purified particles. Another important consideration for getting good transduction efficiency is the cell status. Transduction efficiency varies greatly between healthy cells and unhealthy cells. Therefore, it is essential to keep the cells as healthy as possible. For some cells with high MOI, you could also include additives such as polybrene to enhance the transduction efficiency. However, the overall health of the cells itself is always the most essential element.
7. Does freeze-thaw cycle influence the titer of lentiviruses? Yes, multiple freeze-thaw cycles may reduce the functional titer of the virus stock by up to 2-4 folds. However, first one freeze-thaw cycle does not lower the lentiviral titer. Genemedi recommends our customer not to use lentivirus that has gone through more than two freeze-thaw cycles for titer reason.
8. Why do my cells die in quantity after adding lentivirus to the culture medium? Lentivirus has some level of toxicity to cells. It may cause damage to your cell of interest with either superfluous amounts of lentivirus, or if the infection were allowed to go on for too long a period of time. In these cases, you can adjust the multiplicity of infection (MOI) to a lower range. We recommend replacing the old culture medium with fresh complete medium 4-8 hours post transduction (no later than 12 hours post transduction).
9. What is the capacity of lentiviral vector as an expression system? The cloning capacity for the transgene is approximately 3-4 kb for most vector formats.
10. How do I create a stable cell line using lentivirus? Lentiviruses can stably integrate into the host cell’s genome and obtain a consistent level of expression. With a selectable marker in the lentiviral gene transfer vector plasmid, it is easy to generate a stable cell line using drug selection. You can use qRT-PCR, western blot or other detection methods to estimate the expression level of your gene.
11. How custom lentivirus production service proceeds: If lentiviral construct is ready, please provide us:
1. The construct map or sequence.
2. 1~5 µg of your lentiviral construct plasmid DNA or bacterial culture.
We will prepare Endotoxin-free plasmid and transfect our packaging cell line for your lentivirus production. Lentiviral particles will be harvested followed by concentration and titration.
For 200 ul of lentivirus production, you will receive eight 25 µl aliquots.
If you need us to generate lentiviral transfer vector for your target gene, please:
1. Choose lentiviral vector format from our vector collection and specify the gene of interest.
2. Please send us 1~5 µg plasmid or bacterial culture for your gene if it is available, otherwise please send us cDNA sequence.
We will subclone the gene of interest or shRNA into the chosen lentiviral vector and confirm its accuracy by sequencing.
12. How long does the service take? Is my project too big (or too small)? Get ready-to-transduce, high-quality, high titer lentiviral preparations at the production scale your project needs—even at large scales of up to 10 mL. Use your own lentivector construct or take advantage of our Custom Construct services and we’ll handle vector construction as well. We even have an ultra-high titer offering for demanding applications such as in vivo and stem cell transductions.
13. How are Genemedi lentiviral vectors different from other viral vectors? Genemedi has a particular know-how in the field of lentiviral vector design and production stemming from years of research. This experience has led us to set up optimized protocols and reach high quality standards, constantly improved. Hence, Genemedi lentiviral vector productions show high efficiency, suited for either in vivo, in vitro or transgenesis applications. Our expertise allows us to take on all challenges. Notably, we provide our clients with lentiviral vectors expressing particularly challenging transgenes, for instance, ones that directly interfere with the lentiviral cycle.
14. For what kind of applications can lentiviral vectors be used? Our lentiviral vectors can be used in large array of purposes. Taking advantage of our products can significantly enhance your research. Here are some examples of in vitro and in vivo applications:
a) Stable expression transgenes of interest in target cells.
b) Expression of reporter systems to characterize a given vector in particular contexts.
c) Transient expression of transgenes of interest in dividing cells.
d) Induction of long term expression in non-dividing cells with reduced genotoxicity
e) Generation of cellular models, such as cell lines with recombination induced by transiently expressed genome editing tools (eg: CRISPR)
f) Generation of animal models by zygote injection and transient expression of genome editing tools (KO animals).
15. Which cell type is transduced by Genemedi lentiviral vectors ? Lentiviral vectors are very flexible gene transfer tools and can transduce a large variety of cells. Genemedi can mediate ubiquitous or cell specific expression depending on the envelope and the promoter selected for your vector. Indeed the envelope will determine the entry mechanism of the vector into the cell, and the promoter will define the mechanism of transcription of the transgene. Consequently, the combination of these elements generates ubiquitous transduction and transgene expression in a wide range of cell types.
16. What are the advantages of lentivirus over retrovirus and adenovirus? Lentiviruses comprise a subtype of retroviruses. Lentiviruses can stably integrate into the host genome in dividing, non-dividing and post-mitotic mammalian cells, while retroviruses are less active in this scenario. Adenoviruses can also transduce non-dividing cells, but can’t stably integrate into the host cell’s genome. Adenoviruses also take much more time to design and prepare. In addition, lentiviruses are much less immunogenic than the retroviruses and adenoviruses, making lentivirus more suitable for use in most types of cells and animal models.
17. What are the storage and handling recommendations? Upon receipt, the viral vectors must be stored at -80°C.
The vectors should be taken out from the -80°C freezer at the last moment and kept on ice for gentle thawing. Once thawed, they should be used for transduction as soon as possible to avoid degradation. It is essential to avoid heat shock of the viral vectors and cells. If the vectors should be diluted in medium, use a medium at room temperature to minimize the heat shock experienced by both vectors and cells.
We recommend not refreezing the viral vectors. In order to guarantee viral vectors titer, we recommend keeping freeze-thaw cycles to a minimum. In case that more than one freeze-thaw cycle is required according to your application, Genemedi recommends applying a decrease of about 15% on the titer for each freeze-thaw cycle.
18. What are the safety issues when using Genemedi lentiviral vectors? Genemedi vectors are biosafety level 2 products. The use of retroviral derived vectors implies laboratory biosafety procedures and practices in accordance with regulations applicable in your country. Genemedi vectors are suited for research applications, either in vitro or in vivo, providing that you respect local laws regarding GMO class 2 storage and handling. All Genemedi vectors are distributed for laboratory research use only and cannot under any circumstances be used for diagnostic or treatment applications. For more information on biosafety levels please visit http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
19. What is virus titer? How is lentiviral stock titer determined? In strict sense, the definition of lentiviral vector titer is the number of lentivirus particles required to infect a cell, present in a volume. Titration of lentiviral preparation is important to consistent and reliable experiment result. Two classes of titration methods have been described for evaluation of lentiviral vector titers, including functional and physical titration methods. Functional assay includes determination of transducing units following transducing cells with limiting dilution of lentiviral preparation, assessment of the number of colony forming units following antibiotic selection, as well as FACS when fluorescent reporters are expressed. The latter includes determination of the quantity of p24 antigen by ELISA, reverse transcriptase activity and the genomic RNA concentration in vector preparation. The physical methods provide the information of the number of physical lentivirus particles in lentiviral preparation. Functional assay generates valuable data on the quality of these lentiviral vector particles.
20. How can I increase the transduction efficiency? Try varying particle-to-cell ratio (PPC), incubation volume, temperature and, cell density (if adherent cells are transduced). For adherent cells, we recommend a confluence of about 70%. Following the PPC, adjusting the volume is the next best parameter to change to optimize protein expression.
21. What is the difference between LC3A, LC3B and LC3C, or LC3-I and LC3-II? LC3 is a soluble protein with a molecular mass of ∼17 kDa and is distributed ubiquitously in eukaryotes. It is expressed as the splice variants LC3A, LC3B, and LC3C which display unique tissue distribution. All LC3 isoforms undergo post-translational modifications, especially PE conjugation (lipidation) during autophagy. Upon autophagic signal, the cytosolic form of LC3 (LC3-I) is conjugated to PE to form LC3-PE conjugate (LC3-II), which is recruited to the autophagosomal membranes.
22. Can I use the LV-mRFP-GFP-LC3 products to study autophagy in cells in combination with a lentivirus transfection? Lentivirus transfection may actually induce autophagy, skewing the results. Make certain to include appropriate controls and make sure to leave an appropriate time (48-72hrs) post transfection to lower the basal autophagy level.
23. Will the GFP signal of mRFP-GFP-LC3 that anchored at autophagosomes and fused with lysosome relight after the cell fixed? GFP is no longer delighted reversibly once GFP-LC3 signal was destroyed by the acidic environment.
24. Can transient expression of RFP-GFP-LC3 differ badly from stable expression? Transient transfection of GFP-LC3 can leads to the formation of non-autophagic LC3 puncta due to overexpression. Reports shows that GFP-LC3 is aggregate prone protein. We suggest the use of stable cell line for assessment of GFP-LC3 puncta.
25. Which software do you usually use for counting the number of LC3 dots in the cell? Image J does work. For LC3 puncta, we suggest using the "Watershed" plugin from Image J. But proper threshold adjustment is critical. CellProfiler is another choice. First use the top hat filter. Then with the Identify Primary Objects module, manually adjust the Intensity threshold to optimally select the punctas of the right intensity.
26. How long is chloroquine half life when I treat a cell line for an autophagy study? Chloroquine is an attractive drug agent effective for the treatment of not only malaria but also inhibition of autophagy, which is a promising effect for anti-tumor therapy. Half-life time of this drug is approximately 18 hours in vivo due to the degradation by the liver, but the stability of chloroquine in vitro experiments is expected to be longer. It will depend mainly on the cell line your are working on and their metabolic activity. For example HepG2 cells (human hepatocarcinoma cells) have a strong capacity to metabolize drugs. this might not be the case for other cell line of different tissular origin. I would suggest to add chloroquine when you change your growth medium.
27. What medium is best to use to induce LC3 puncta in HeLa cells? We recommend the media used by Axe et al. (2008) JCB 182: 685-701. The recipe is 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 20 mM Hepes, pH 7.4, Add 1% BSA and pass through a 20um filter before use.
28. What is the best applicable inhibitor of autophagy? I prefer to use CQ (chloroquine) in all of my researches about autophagy inhibition. The using of CQ is quite easy, since it is easy dissolve in water (unlike 3-MA in DMSO). One time I have been used 3-MA, actually I did not like to work with it. Bafilomycin is another choice for you.
29. When to add bafilomycin to study autophagy? it is better to do a time course to be sure. We use at least two times (2 and 4 hours) to starved the cells and we add baf for 2 hours (for 4 hours: the 2 last hours). We then follow LC3-II and P62 by WB. To see the degradation of p62, sommetime 4 hours is better than 2hours.
30. Which neuronal cell line should I use for autophagy level? SHSY5Y and PC12 are two well established neuronal cell lines, human and rat cell lines, respectively. You may also try Neuro2A, it is a mouse cell line.
31. How to distinguish selective and non-selective autophagy? You can analyze selective mitophagy by interaction/co-localization study of mitochondrial marker protein and the autophagy specific adaptor protein LC3II.
32. When to Add the autophagy treament in cell culture models? First, some cells are very sensitive to these inhibitors and you need to optimize your conditions. The second point, if you like to stop autophagy process after inducing it to compare whether it is a cell death or cell survival you may add it just hours (2-3) before treatment. However, many articles prefer to use it as pre-treatment 1 hour before any other treatment.
33. What is “CRISPR”? “CRISPR” stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. In the field of genome engineering, the term “CRISPR” or “CRISPR-Cas9” is often used loosely to refer to the various CRISPR-Cas9 and -CPF1, (and other) systems that can be programmed to target specific stretches of genetic code and to edit DNA at precise locations, as well as for other purposes, such as for new diagnostic tools.
34. How does the CRISPR/Cas9 system work? CRISPR “spacer” sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system – binds to the DNA and cuts it, shutting the targeted gene off. Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA. These techniques allow researchers to study the gene’s function.
35. How does CRISPR-Cas9 compare to other genome editing tools? CRISPR-Cas9 is proving to be an efficient and customizable alternative to other existing genome editing tools. Since the CRISPR-Cas9 system itself is capable of cutting DNA strands, CRISPRs do not need to be paired with separate cleaving enzymes as other tools do. They can also easily be matched with tailor-made “guide” RNA (gRNA) sequences designed to lead them to their DNA targets. Tens of thousands of such gRNA sequences have already been created and are available to the research community. CRISPR-Cas9 can also be used to target multiple genes simultaneously, which is another advantage that sets it apart from other gene-editing tools.
36. How does CRISPR-Cpf1 differ from CRISPR-Cas9? CRISPR-Cpf1 differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics.
First, in its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.
Second, and perhaps most significantly, Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving ‘blunt ends’ that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.
Third, Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be re-cut, allowing multiple opportunities for correct editing to occur.
Fourth, the Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting, for example, the malaria parasite genome and even the human genome.
37. What other scientific uses might CRISPR have beyond genome editing? CRISPR genome editing allows scientists to quickly create cell and animal models, which researchers can use to accelerate research into diseases such as cancer and mental illness. In addition, CRISPR is now being developed as a rapid diagnostic. To help encourage this type of research worldwide, Feng Zhang and his team have trained thousands of researchers in the use of CRISPR genome editing technology through direct education and by sharing more than 40,000 CRISPR
38. How to design the 20bp target-specific sequence? The 20bp target-specific sequence should precede NGG (PAM). Please BLAST the seed region (8-14 bp PAM-proximal) of the 20bp target sequence to make sure it’s unique along the genome to guarantee its specificity.
39. How to avoid off target issue using CRISPR/Cas? You can blast your target sequences. If the off-target sequences don’t have the PAM (NGG), then they won’t be targeted by CRISPR/Cas9. You also want to choose target sequences with mismatches in the 8-14 bp at the 3’ end of the target sequences. This way, the off-target issue can be decreased dramatically. For therapeutic purpose, you can use Cas9 nickase which only cuts one strand.
40. How many target RNA sequences should I use for a genome editing project? Due to the un-predicable nature of gRNA, we recommend 3 and more gRNA targeting sequences to be designed to make sure that at least one targeting sequence will provide efficient cleavage.
41. Do you know the specific cleavage site of the Cas:gRNA complex in terms of where in the targeting sequence the cleavage occurs? Cas9 cleaves at 3 bp away from the 3’ end of the target sequence in the genome.
42. Why I cannot find the gRNA targeting sequences in the cDNA sequence? The targeting sequences could be located in either exon or intron in the genome; the cDNA sequences only contain the exons. CRISPR/Cas9 will target the genomic sequence, then genome editing will be achieved.
43. The transfection efficiency of my cell line is only 20%, how to enrich CRISPR transfected cells? You can use pCas-Guide-EF1a-GFP to enrich transfected cells since GFP is also expressed. We also have pCas-Guide-EF1a-CD4 vector; you can use anti-CD4 antibody beads to enrich transfected cells. Alternatively, you can transfect a plasmid with a selection marker and select the cells.
44. How to screen the edited cells after transfecting the CRISPR/Cas9 vector? For mutations, you can do genomic PCR and sequence it. If you do gene knockout, the selection marker in the donor template DNA will help the selection. If no donor DNA for gene knockout out, then genomic PCR and sequencing to confirm indels. If necessary, you can isolate individual cell colonies for introduction of specific mutations and other genome editing applications. You can do WB for gene knockout after isolating single cell colonies.
45. Does CRISPR/Cas system work for non-dividing cells? NHEJ repair works in non-dividing cells; HDR is not active in non-dividing cells.
46. Using CRISPR, can you get monoallelic knockout (heterozygous) or biallelic knockout (homozygous)? CRISPR/Cas9 double-strand cleavage is very efficient. If just using CRISPR/Cas9 vectors to introduce indels, if transfection efficiency is high, more biallelic knockout can occur. In the presence of donor DNA, since homologous recombination may be a limiting factor, some cells contain monoallelic knockout and some cells contain biallelic knock out.
47. If I want to use CRISPR/Cas9 to knock down a certain gene, what kind of negative control should I use? You can use a scramble control, pCas-Scramble, SKU GE100003, or pCas-Scramble-EF1a-GFP, SKU GE100021.
48. For gene targeting in mice, do you recommend transfecting ES cells or pronuclei? You can do both. You can inject mRNA (gRNA and Cas9 mRNA) or plasmid DNA (target sequence cloned pCas-Guide) into the zygotes or ES cells.
49. How do you make sure that Cas9 will not integrate in genome if you use lentivector? For screening purpose, for short term, integration of Cas9 into the genome for 2 weeks does not affect cells.
50. Do you see variability in success with different cell lines? Yes, depending on the cell line and the gRNA sequences.
51. What is the known CRISPR/Cas9 editing efficiency relative to other genome editing approaches? In general, the genome editing efficiency of CRISPR/Cas9 is similar or higher than TALEN. However, CRISPR/Cas9 is much more simple and easy to do. You will need to engineer the protein to recognize new DNA sequence in TALEN system, while CRISPR/Cas9 is RNA based.
52. Is there any safety issue with this pLenti vector? The pLenti vector is a third generation lentiviral vector and it is the safest lentiviral vector because both LTRs are truncated. Please contact the biosafety office at your institution prior to use of the pLenti vector for permission and for further institution-specific instructions. BL2/(+) conditions should be used at all times when handling lentivirus. All decontamination steps should be performed using 70% ethanol/1% SDS. Gloves should be worn at all times when handling lentiviral preparations, transfected cells or the combined transfection reagent and lentiviral DNA.
53. Do I get monoallele knock-out or biallele knock-out using the homology-mediated knock-out kit via CRISPR? What do I need to do to get biallele knock-out? If you isolate single cell colonies, in some cells gene knock-out may occur only in one allele; in some cells gene knock-out may occur in both alleles. If you only have monoallelic knockout and you want to get biallelic knockout, you can order another donor vector containing a different mammalian selection marker, such as blastocidin or neomycin resistant marker. Make sure the other allele is intact as it can be targeted and repaired via NHEJ; confirm with genomic PCR and sequencing. You can do the knockout procedure again with the new donor vector to target the second allele (one allele is already targeted and replaced with GFP-puro cassette).
54. What could be the reason that I couldn’t get my gene of interest knocked out? If your target gene is essential for cell survival, you might not be able to get constitutive gene knockout. Conditional gene knockout may be needed.
55. Can I use the nickase instead of wild-type Cas9? Yes. We have the reagents for the Cas9 D10A nickase, and have successfully tested our double nickase designs. However, in order to create mutagenic DSBs, the nickase requires the correct targeting of two appropriately-spaced sgRNAs on opposite strands, flanking the break site. Because proper sgRNA targeting requires the presence of the N-G-G “PAM” site immediately following the recognition site, it might not always be possible to use the nickase for DSB formation. There are also “high-fidelity” variants of Cas9 nuclease that edit genes with greater specificity than wild type Cas9, but sometimes with reduced efficacy and with increased design constraints. However, since these high fidelity variants use only one sgRNA, they are easier to work with than Cas9 niclases.
AAV(Adeno-Associated Virus) vector system