Newsletter Techniques
March 2012 - Your Top 10 Lentivector Questions (See full March 2012 newsletter)
December 2011 - A TALe to Remember (See full December 2011 newsletter)
September 2011 - How to create your perfect MCS (See full September 2011 newsletter)
March 2012: Your Top 10 Lentivector Questions
Trying to find a lentiviral plasmid for your experiment? See our lentiviral reference page.
Or contact us at [email protected]
At Addgene, we receive questions daily from trained molecular biologists, along with scientists taking their first plunge into the world of recombinant DNA. By far, the topic most inquired about over the past 8 years has been lentiviral plasmids. For good reason, in the past decade lentiviral vectors have in many ways changed the way all types of researchers do science. Here at Addgene, we’ve sent out over 30,000 lentivectors since 2004 and in February alone we distributed over 800(!). We've assembled some of our most common FAQs below for your reference.
What is a lentivirus? How is it different than a retrovirus?
Lentiviruses are a subtype of retrovirus. The main difference between lentiviruses and standard retroviruses from an experimental standpoint is lentiviruses are capable of infecting non-dividing and actively dividing cell types, whereas standard retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses. Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging. However, the isoforms of these proteins used by different retroviruses and lentiviruses are different and lentiviral vectors may not be efficiently packaged by retroviral packaging systems and vice versa.
What is a lentivector? Are there different types?
Lentivectors or lentiviral plasmids are based off the genomes of lentiviruses. For producing lentiviral particles, you typically need three plasmid components (as illustrated in the diagram below):
- A lentiviral vector or “transfer vector” containing the shRNA or transgene and the flanking LTRs
- A packaging vector or set of packaging plasmids
- An envelope vector
The transfer plasmid is often the one which requires the most consideration when deciding what to use for your experiment. Often the envelope and packaging plasmids are interchangeable with many different transfer plasmids.
What is the difference between a 2nd and 3rd generation lentivector? How can I tell the difference?
For a full description of 2nd and 3rd generation lentiviruses please see our page on packaging plasmids. Briefly, 2nd generation lentiviral systems use more viral proteins (on fewer plasmids) in order to produce functional lentiviral particles than 3rd generation systems.- 2nd generation packaging systems – express the HIV gag, pol, rev, and tat genes all from a single packaging vector such as psPAX2
- 3rd generation packaging systems – express gag and pol from one packaging vector and rev from another, such as pMDLg/pRRE and pRSV-Rev. 3rd generation packaging systems DO NOT express tat. 3rd generation lentiviral systems are considered safer than second generation systems, but are more difficult to use because they require transfection with four separate vectors in order to create functional lentivirus.
IMPORTANT: A 3rd generation transfer vector can be used with a 2 nd generation packaging system, but a 2 nd generation transfer vector cannot be used with a 3rd generation packaging system.
Can I package my lentiviral vector with commercial packaging cell lines?
It is possible, although the highest titers are usually obtained with using the standard 293T cell line. 293T cells need to be carefully passaged and cared for. Although they may seem pretty robust, it's recommended to transfect cells that have only gone through one or two passages.
Can lentivectors be used in direct transfections without producing virus?
Lentiviral transfer vectors can be used in transient transfections to achieve expression of the transgene. Lentiviral transfer vectors are not designed specifically for transient transfections, therefore there may be consequences on transgene expression due to the lentiviral LTRs. It is not explicitly recommended that you use lentiviral transfer vectors for simple transfections, but it is possible.
How can I tell if a lentivector is supposed to be used to express cDNAs or shRNAs?
An RNA Pol II promoter, such as CMV or RSV, is required to drive expression of your cDNA. In contrast, an RNA Pol III promoter, such as U6 or H1, is needed for shRNA expression. You can definitely swap out promoters if you've found a useful transfer plasmid but it doesn't have the right promoter for your experiment.
What dictates lentiviral host cell range (tropism)?
Lentiviral tropism is determined by the ability of the viral envelope protein to interact with receptors at the host cell surface. The VSV-G envelope protein is commonly used in lentiviral particle production because it confers broad tropism over a range of species and cell types. For more information on different envelopes and their tropism check out this reference.
Are lentivectors safe to use in the lab?
While we don't work with the viruses themselves at Addgene, labs around the world use lentiviral plasmids on a daily basis. Lots of advances have been made to make the plasmids safer over the years. BL2 in the US and its equivalent in Europe BSL-2, are appropriate for most uses of lentiviral vectors, but bio-safety should always be considered with respect to the precise nature of experiments being performed.
The NIH provides basic guidelines to ensure that research is being performed within the highest standards possible to protect researchers. Visit the NIH's website for more details about safety considerations. Specific questions about how biosafety relates to your research should be directed to your institution’s Biosafety Committee.
Will I get a larger titer with 2nd or 3rd generation packaging systems?
Second generation systems will always produce a greater titer because they involve transfecting fewer plasmids (3) versus the third generation system which involves using 4 plasmids to generate virus.
What plasmids does Addgene have for lentiviral expression and production?
Addgene has over 8000 lentiviral plasmids. Check out some of our most popular lentiviral plasmids here.
December 2011: A TALe to Remember
Imagine being able to manipulate a specific region of DNA in the genome of your favorite species almost as easily as correcting a typo in the latest draft of your dissertation. Well, that will probably never happen. Yet with transcription activator-like (TAL) effectors, scientists in several biological disciplines are coming closer to this DNA targeting ideal.
For over ten years, zinc finger (ZF) arrays have been the go-to technology for targeting enzymes and other useful protein domains to a specific DNA sequence ( Fig. 1A ). Scientists have identified a large number of zinc fingers that recognize various nucleotide triplets. With some trial and error, ZF arrays are able to recognize their targets with fairly high specificity. Fifteen years of research has shown that when fused to a nuclease or activation domain, a ZF array is an effective targeting mechanism for molecular tools. ZFs are not without their limitations, however. Not every nucleotide triplet has a corresponding zinc finger, and interactions between zinc fingers within an array can reduce their specificity.
Two years ago, groups led by Jens Boch at the Martin-Luther-University Halle-Wittenberg and Adam Bogdanove at Iowa State University published the nucleotide recognition code of the TAL effectors, which were isolated from the plant bacterial pathogen Xanthomonas. The central TAL targeting domain is composed of 33-35 amino acid repeats. These repeats only differ from each other by two amino acids, their repeat-variable di-residue (RVD). Ultimately, it is this RVD that determines which single nucleotide the TAL effector will recognize: ( Fig. 1B ) HD targets cytosine, NI targets adenenine, NG targets thymine, and NN targets guanine (though NN can also bind adenenine with lower specificity).
With the template for studying this type of technology already laid by the zinc finger community (see interview with Adam Bogdanove), progress on TALe research has been swift. The incentives are high. Since ZF targets are confined to sequences composed of triplets with corresponding zinc fingers, potential targetable sites in your average genome are every 500 bp. TAL effectors have some restrictions (for example, the target must start with a T), but they still have potential targetable sites approximately every 35bp. Researchers are still determining the importance of context for each TAL effector within an array, but early studies suggest the likelihood of context having a negative effect on specificity is on par with ZF arrays, if not slightly lower.
Perhaps one of the most appealing features of the TAL effector arrays is the ease with which they can be made. Intuitively, one would expect that assembling relatively small, repeat-laden DNA regions into a single construct would be technically challenging. Thanks to efforts by the Bogdanove group and Daniel Voytas’s group from the University of Minnesota, arrays can be assembled in a matter of days. In an open access Nucleic Acids Research article that came out in the spring of 2011, the groups describe a set of customized plasmids that can be used with the recently developed Golden Gate cloning method to assemble “multiple DNA fragments in an ordered fashion in a single reaction.” Using these plasmids, which are available as a kit from Addgene, custom arrays consisting of 12-31 repeats can be assembled and inserted into a variety of backbones in just a few steps ( Fig. 2 ). The Bogdanove group also hosts web-based software to help scientists find potential TAL effector targets. The site, which will be updated soon with more features, is already receiving hundreds of unique visitors every month.
The Voytas/Bogdanove TALEN kit came online in the summer of 2011 and has already become the best-selling kit in Addgene’s history. Dr. Keith Joung’s lab at Massachusetts General Hospital also recently released a TALEN kit through Addgene. This kit, first described in Nature Biotechnology in August, uses a serial ligation protocol to assemble arrays. Dr. Joung, co-founder of the Zinc Finger Consortium with Dr. Voytas, has considerable experience in the DNA targeting field. A third kit, a “TALe Toolbox”, from Dr. Feng Zhang’s group at the Broad Institute, will be available from Addgene before the end of the year. Dr. Zhang ‘s lab already published a paper on TAL effectors in Nature Biotechnology earlier in 2011 and will describe this new kit for assembling TAL effector arrays in a forthcoming issue of Nature Protocols (in press). The Zhang lab also hosts a helpful website that provides several TAL effector resources: http://taleffectors.com/
Whether you work in a C. elegans lab and have been struggling to mutate a specific gene or your anxiously investigating gene therapy options for a rare disease in humans, TAL effectors could become a key tool in your plasmid toolbox. Addgene has rapidly become an important resource for this new technology, and we hope to continue to have a strong scientific partnership with both the labs that continue to hone these tools and the researchers who are learning to wield them. Learn more about Addgene’s TAL effector and TALEN kits at www.addgene.org/talen/
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. Nucleic Acids Res. 2011. Jul;39(12):e82. Epub 2011 Apr 14.PubMed.
Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR. Nature Biotechnology 2011 Aug 5;29(8):697-8. doi: 10.1038/nbt.1934. PubMed.
TAL effectors: customizable proteins for DNA targeting. Bogdanove AJ, Voytas DF. Science .2011 Sep 30;333(6051):1843-6. PubMed.
Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P. Nature Biotechnology 2011. 29, 149-153. PubMed.
TALEs for the masses. Rusk N. Nat. Mthods. 2011 Mar;8(3):197. PubMed.
Move over ZFNs. Nature Biotechnology .2011. Aug 5;29(8):681-4. doi: 10.1038/nbt.1935. PubMed.
See the Q&A with Dr. Adam Bogdanove and his thoughts on the TALe technology here.
September 2011: How to create your perfect MCS
Does this scenario sound familiar - you desperately need to make retrovirus expressing your new favorite gene and the only restriction sites in your favorite vector are also in the coding sequence of your gene? You can remove them by site directed mutagenesis, or settle for a different vector, but why not just improve the multiple cloning site (MCS) of your favorite vector. It is a couple days of work that will pay off for years to come.
The following technique is one way to easily improve the MCS of any vector. Although we will discuss how to add new restriction sites, it is just as easy to add short tags (like HA or FLAG) using this method. For the purposes of this tutorial we will show you how easy it would be to take the very popular retroviral vector pBabe-puro (Addgene plasmid #1764 ) which has a relatively limited MCS (BamHI - SnaBI - EcoRI - SalI) and expand that with the addition of NdeI, PacI, AscI, and MfeI to drastically improve the MCS - and best of all - for maximal compatibility they all work in NEB buffer 4.
Briefly, we will design overlapping oligos that once annealed can be cloned directly into the overhangs generated by restriction digest of existing sites in the original vector.
Designing oligos: We want to add NdeI, PacI, AscI and MfeI sites so we will be generating a top oligo with each of these sequences in tandem (NdeI - CATATG - PacI - TTAATTAA - AscI - GGCGCGCC - MfeI - CAATTG) with the bottom oligo being the reverse compliment so that they will anneal.
Top: 5'-CATATGTTAATTAAGGCGCGCCCAATTG-3' = 28bp
Bottom: 3'-GTATACAATTAATTCCGCGCGGGTTAAC-5'
We also need to make sure to include the additional bases necessary to compliment the overhangs generated when digesting the vector with EcoRI and SalI (see diagram). To do this we need to add 5'-AATTC and G-3' to the top oligo and 3'-G and CAGCT-5' to the bottom oligo making our final oligos 34bp each.
Top: 5'-AATTCCATATGTTAATTAAGGCGCGCCCAATTGG-3'
Bottom: 5'-TCGACCAATTGGGCGCGCCTTAATTAACATATGG-3'
Order from your favorite oligo synthesis company. NOTE - if you phosphatase treat your cut vector it is necessary to use 5'-phosphorylated oligos. This is an option that can be added when ordering them or can be performed enzymatically later.
Digest and purify vector: While waiting for your oligos to arrive, digest 1ug empty pBabe-puro with EcoRI and SalI and gel purify.
Anneal oligos: The oligos should be resuspended in annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1m EDTA) and mixed in equimolar concentrations. We recommend mixing 2ug each in a total volume of 50uL - add additional annealing buffer if necessary to get to 50uL. Efficient annealing can be achieved by one of two methods:
place the mixed oligos in a 1.5mL microfuge tube
- place tube in 90-95 degree hot block and leave for 3-5 minutes
- remove the hot block from the heat source (turn off or move block to bench top) allowing for slow cooling to room temperature (~45 minutes).
place mixed oligos in a PCR tube, and place tube in a thermocycler programmed to start at 95 degrees for 2 minutes and then to gradually cool to 25 degrees over 45 minutes.
Ligation: Dilute 5uL of annealed oligos with 45uL nuclease-free water and quantify (should be about 8ng/ul). Mix the annealed oligos with cut vector in ratios between 4:3 and 6:1 (ie 100ng vector with between 75-600ng annealed oligos) in a standard ligation reaction.
Transform 2-3uL into your favorite bacteria and plate. Be sure to pick multiple colonies for mini-prepping and verify insert by sequencing. And don't forget, if you generate a useful plasmid; share it with the rest of the scientific community by depositing it at Addgene.
