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查看更多产品信息 293A Cell Line - FAQs (R70507)
17 个常见问题解答
大多数腺病毒包含在漂浮的细胞中，细胞破裂之前它们并不会释放到培养基中。我们推荐每三天左右更换一次培养基，直至大量细胞变大变圆，并从塑料培养皿上脱离下来。一旦细胞发生破裂，游离的病毒就会迅速感染邻近的细胞。如果您很担心在更换培养基的过程中损失这些受到感染的细胞（及内在的病毒），您可保留含有漂浮细胞的培养基，冻/融三次后将其中的一小部分（可能1/10左右）与新鲜培养基一道重新加回新鲜培养基中。或者，您也可使用新鲜培养基，以显著高于每三天一次的频率进行半换液。
任何293来源的细胞系及其他表达E1蛋白的细胞系均适用于腺病毒的生产。293A中A代表“贴壁”，是因为293A细胞（只是常规293细胞的一个单细胞克隆）具有贴壁的倾向，在组织培养皿中会形成平铺生长良好的单层细胞。这就是为什么这些细胞很适合应用于噬斑测定实验。普通的293细胞不会形成这样的单层细胞；它们生长所形成的细胞层会出现孔洞和空白之处。
我们使用经测试不含支原体的Gibco FBS（货号16000-044），并用下列塑料器皿进行293A细胞的培养：
T175—Fisher 货号 10-126-13；这是一款Falcon培养瓶，带有0.2 μm透气孔的密封盖。
T75—Fisher 货号 07-200-68；这是一款Costar培养瓶，带有0.2 μm透气孔的密封盖。
100 mm 平板—Fisher 货号 08-772E；这是一款针对组织培养应用进行预先处理的的Falcon聚苯乙烯平板。
我们在日常的细胞培养/维持过程（除在293A细胞中生成腺病毒时的细胞裂解过程）中使用这些平板获得了绝佳的贴壁效果。
在将表达克隆转染进293A细胞之前，您须暴露载体上左侧和右侧的病毒反向末端重复序列（LTRs），以辅助病毒复制和包装过程的顺利进行。同时，这一操作也将去除细菌序列（即pUC来源和氨苄青霉素抗性基因）。pAd/CMV/V5-DEST和pAd/PL-DEST载体均包含Pac I限制性酶切位点（请分别参见使用手册第20页和第22页图谱中的Pac I位点）。
注意：请确保你的目的DNA序列中不含任何Pac I限制性酶切位点。如果您无法应用Pac I位点进行处理，您也可换用Swa I位点。
Most of the adenovirus is contained within the floating cells and is not released into the medium until those cells burst. We recommend changing the medium every 3 days or so until it is obvious that a lot of cells become big and rounded and are detaching from the plastic. Once a cell bursts, the free viruses rapidly infect the neighboring cells. If you're ever worried that you're losing infected cells (and therefore potential virus) in your medium changes, you can always save the medium with the floating cells, freeze/thaw it 3 times and then use a little (maybe 1/10th) and add it back to your culture with fresh media. Or, replace only half of the medium with fresh medium and do this more often than every three days.
Any 293-derived cell line or other cell line that expresses the E1 proteins may be used to produce adenovirus. In 293A cells (recommended for adenovirus production), "A" stands for "adherent" because the 293A cells (which are just a single-cell clone of regular 293) tend to adhere and form nice flat monolayers in tissue culture dishes. This is why they work so well for plaque assays. Regular 293 cells will not form the same type of monolayers; they exhibit holes and gaps during growth.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) and use the following plasticware for 293A cells:
T175: Fisher Cat. No. 10-126-13; this is a Falcon flask with a 0.2 µm vented plug seal cap.
T75: Fisher Cat. No. 07-200-68; this is a Costar flask with a 0.2 µm vented seal cap.
100 mm plate: Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate.
We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).
Before you can transfect your expression clone into 293A cells, you must expose the left and right viral inverted terminal repeats (ITRs) on the vector to allow proper viral replication and packaging. This also removes bacterial sequences (i.e., pUC origin and ampicillin resistance gene). Both pAd/CMV/V5-DEST and pAd/PL-DEST ;vectors contain Pac I restriction sites (see maps on pages 20 and 22 of the manual (http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf), respectively, for the location of the Pac I sites).
Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites. If you are unable to use the Pac I site, you can use the Swa I site.
No. The ViraPower system uses adenovirus type 5. Adenoviruses (Adenoviridae) and adeno-associated viruses (Parvoviridae) are completely different. Adeno-associated viruses are often associated with adenovirus infections, hence the name. Since they are thought to be virtually non-pathogenic, they are attractive vectors for gene therapy. The disadvantage is that they can package only about half the foreign DNA that adenoviruses can.
Clone your gene of interest into the pAd/CMV/V5-DEST (or pAd-PL-DEST if you want to use your own promoter). Prior to cloning, if desired, propagate this vector in One Shot ccdB Survival 2 T1R Competent Cells (Cat. No. A10460) as described below. After cloning your gene of interest, propagate in E. coli strain TOP10. pAd/CMV/V5-GW/lacZ is provided as a positive control vector for expression.
Digest recombinant plasmid with Pac I to expose the ITRs (inverted terminal repeats).
Transfect (we recommend Lipofectamine 2000 reagent) E1-containing cells (293A cells) with linear DNA (only 10% of transfected cells will make virus).
Infected cells will ball up, and release virus to surrounding cells, which in turn will be killed and ball up. Look for plaques in the monolayer created by areas cleared by detaching, balled up cells (it takes 8-10 days to see visible plaques from this initial transfection).
Collect a crude viral lysate.
Amplify the adenovirus by infecting 293A producer cells with the crude viral lysate. Harvest virus after 2-3 days when cells ball up. Determine the titer of the adenoviral stock by performing a plaque assay. The virus generated is adenovirus type 5 (subclass C).
Add the viral supernatant to your mammalian cell line of interest to transduce cells.
Assay for recombinant protein of interest.
Once you have your gene of interest in the adenoviral vector, you can simply re-amplify when you need more of the virus. You do not need to repeat cloning steps and transfections each time.
When cloning or propagating DNA with unstable inserts (such as lentiviral DNA containing direct repeats), we recommend using the following modifications to reduce the chance of recombination between direct repeats:
- Select and culture transformants at 25-30 degrees C.
- Do not use "rich" bacterial media as they tend to give rise to a greater number of unwanted recombinants.
-If your plasmid confers chloramphenicol resistance, select and culture transformants using LB medium containing 15-30 µg/mL chloramphenicol in addition to the antibiotic appropriate for selection of your plasmid.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Ultracentrifugation is the most commonly used approach and is typically very successful (see Burns et al. (1993) Proc Natl Acad Sci USA 90:8033-8037; Reiser (2000) Gene Ther 7:910-913). Others have used PEG precipitation. Some purification methods are covered by patents issued to the University of California and Chiron.
Adenovirus is concentrated using CsCl density gradient centrifugation (there is a reference for this procedure in our adenovirus manual) or commercially available columns.
This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) without any modifications. We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources.
We use the following plasticware for 293A and 293FT cells:
T175--Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 µm vented plug seal cap.
T75--Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 µm vented seal cap.
100 mm plate--Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate
We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).
Viral vectors:
Store lentiviral and adenoviral expression vectors (plasmid DNA) at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely. Glycerol stocks of vectors transformed into bacteria should always be stored at -80 degrees C.
Virus:
Both adenovirus and lentivirus particles should be aliquoted immediately after production and stored at -80 degrees C.
Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.
Adenovirus particles can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.
When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Thermo Fisher Scientific has also engineered specific safety features into the lentiviral system.
Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If you're interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO (D-TOPO) and Gateway version of the kit to provide flexibility in the cloning of the gene of interest.
If you're looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.
For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST vector: 6 kb
pLenti6/V5-DEST vector: 6 kb
pLenti6/V5/D-TOPO vector: 6 kb
pLenti6/UbC/V5-DEST vector: 5.6 kb
For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST vector: 6 kb
pAd/PL-DEST vector: 7.5 kb