|
|
Na2HPO4.2H2O, 8.6 g/L
KH2PO4, 3g/L
NH4Cl, 1 g/L
NaCl, 0.5 g/L
glucose, 2 g/L
glycerol, 10 g/L
yeast extract, 10 g/L
MgSO4, 2.5 mM
Fe(III)-citrate, 250 M
H3BO3, 49 μM
MnCl2, 79 μM
EDTA, 23 μM
CuCl2, 9 μM
Na2MoO4, 10 μM
CoCl2, 11 μM
Zn-acetate, 36 μM.
1. On the day before titering, split each 100 mm plate of 293 cells to three 60 mm dishes. The number of dishes prepared should be six per virus stock to be titered (two for each dilution to be assayed). Care should be taken to prevent clumps of cells in the monolayer by vigorously dispensing the cell suspension. The dishes should be just confluent when used.
2. On the day of the titering, thaw the virus stock to be titered, Prepare serial 10- or 100-fold dilutions of the virus stocks to be titred in DMEM, 2% FBS and Penicilliiun/streptomycin in 6 ml polystyrene culture tubes in a final volume of 2.0 ml.
3. Aspirate the growth medium from the monolayers of 293 cells.
4. Overlay two 60 mm plates of 293 cells with 0.5 ml of each prepared virus dilution. Record on each dish details of the virus and the dilution of stock.
5. Incubate the cells at 37 oC for 1 hour, rocking at 15 min intervals.
6. Melt 1.3% Noble agar in a microwave and place in a 45 oC water-bath for it to equilibrate. Place 2X MEM supplemented with 4% FBS in a 37 oC water bath. Immediately before using, add an equal volume of the molten 1.3% Noble agar to the 2 X MEM.
7. Aspirate infectious virus medium from the cells.
8. Overlay the cells with 6.0 ml of the Noble agar/MEM, 2% FBS. This must be done very gently so as not to disturb the cell monolayer; 293 cells are not strongly adherent, so care is important. Add the agar preparation slowly to the side of the dish. Rock or swirl the dish very gently tp cover the cells and mix in any remaining trace of liquid DMEM. Allow the overlaid cultures to stand at room temperature until the agar solidifies.
9. Incubate the infected plates at 37oC. Plaques should begin to be visible within about seven days.
10. Count the plaques. Do this as follows:
a) Observe the plates against a dark background, or against fluorescent lighting, whichever makes the plaques most readily visible/ S,a;; plaques will appear as white pin-points on the relatively clear background. As the plaques grow, they will appear as larger white spots on the cell lawn and later will form white rings. Cell clumps look macroscopically like plaques at first, but will not grow. Microscopically, plaques are comprised of rounded up cells areas on the monolayer, whereas cell clumps look just as their name suggests as a very dense, raised area of cells.
b) Mark the location of visible plaques with a felt-tipped pen.
c) Count the plaques over a period of several days until the number remains constant.
11. to determine the titer, multiply the number of plaques counted by the dilution factor. Average the results of the serial dilutions.
for example, 1 ul of stock, diluted to 1 ml equals 1:1000 dilution. Infecting with 0.5 ml of the 1:1000 dilution results in a final dilution factor of 1:2000. For large scale purified virus preparations, assaying dilutions of 10^-18, 10^-9 and 10^-10 generally provides an appropriate range.
|
Reagents |
Volume/mass |
Final concentration |
|
Acid Phenol |
380mL |
38% |
|
Guanidine thiocyanate |
94.53g |
0.8M |
|
Ammonium thiocyanate |
30.45g |
0.4M |
|
Sodium acetate, pH 5.0 |
33.4mL of 3M stock |
0.1M |
|
Glycerol |
50mL |
5% |
|
RNase-free water |
Adjust final volume to 1L |
|
So called Solution D (based on Chomczynski and Sacchi1987/2006) is:
Prepare stock with:
(stored <3 months at room temperature)
Working solution from stock:
(store <1 month at RT)
Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):
|
The year 1953 saw the isolation of the Adenovirus which was soon recognized as an invaluable tool for investigating mammalian molecular biology. Several of the distinguished features of Adenovirus have made it the preferred vehicle for gene transfer and transgene expression in mammalian cells. The following presents a small overview of the biology of Adenovirus. Pathology Adenoviruses are associated with a number of disorders (eg. common cold), most of which are mild. The pathology is primarily from inflammation and loss of infected epithelial cells. Viruses of subgroup C (serotype 2, 5) cause various respiration infections in confined groups (elderly, military recruits and children). Genome Adenovirus is a non-enveloped 80-110 nm diameter virus presenting an icosahedral symmetry. Human Adenoviruses contain a linear, double stranded DNA genome, with a terminal protein (TP) attached covalently to the 5′ termini. The DNA, which has a length of approximately 36,000 bp, is wrapped in a histone-like protein and has inverted terminal repeats (ITRs) of 50-200 bp, which act as origins of replication. Structure The hexon, penton base, and knobbed fiber are the most important capsid proteins with regards to gene delivery. Hexon is the major protein forming the 20 triangular faces of the viral capsid. The 240 hexon capsomers in the capsid are trimers, each interacting with six other trimers. The 12 vertices are formed by the penton capsomere, a complex of five copies of the penton base, and three copies of fiber. Each penton capsomere interacts with five hexon capsomeres, one from each of the five faces that converge at the vertex. The knobbed fiber protrudes from the fiber base.
Figure 1: Adsorption and entry into the cell The adsorption of the virus to target cell receptors involves high-affinity binding via the knob portion of the fibre. The prime receptor for human Adenovirus serotype 5 is identical to that for coxsackie B virus and has been named the Coxsackie/Adenovirus receptor (CAR). After the attachment step, interaction between the penton base and av integrins on the cell surface leads to internalisation of the virus through endocytosis. Once inside the cell, the virus escapes the endosome with help of the penton base, and translocates to the nuclear pore complex, where the viral DNA is released into the nucleus and transcription begins. Transcription, replication and viral packaging take place in the nucleus of the infected cell.
Figure 2: Transcription A complex series of splicing accompanies transcription, and genes are transcribed from both strands. Adenovirus transcription is a two-phase event, early and late, occurring before and after viral DNA replication, respectively. The early transcribed regions are E1, E2, E3 and E4. The E1 gene products can be further subdivided into E1A and E1B. E1 gene products are involved in the replication of the virus. The E2 region is subdivided into E2A and E2B. These proteins provide the machinery for viral DNA replication and the ensuing transcription of late genes. Most of the E3 proteins are involved in modulating the immune response of infected cells, a function not essential for viral growth in vitro. The gene products encoded by the E4 region (called ORFs 1-6/7) are involved in the metabolism of virus messenger RNA and provide functions that promote virus DNA replication and shut-off of host protein synthesis. Furthermore, E4 products prevent viral DNA concatenation. Recombinant Adenovirus Adenovirus has been adapted so that it can be used as both gene delivery and gene therapy tools. Recombinant Adenovirus (rAd) generated with the technologies offered by Qbiogene have their E1 and E3 regions deleted. The E1 deletion prevents the recombinant Adenovirus from replicating and therefore no cell lysis occurs. Once packaged into a complementing cell line, i.e. a cell line that provides the E1 products in trans (e.g. QBI-HEK 293A cells), viral replication is made possible. The E3 region, not essential for viral growth, is also deleted. These two deletions allow the introduction of the transgene of interest into the virus. In addition to these two deletions, Qbiogene offers custom services with protease (PS) deleted recombinant Adenovirus. The main components of the PS deleted system are also available under a license agreement. For more info on PS deleted Adenovirus, please refer to the MERLIN® Custom Services section of this catalog. |
Gene delivery, gene therapy and protein over-expression with recombinant Adenovirus Gene delivery consists in introducing DNA and RNA into cells, tissues, or organisms, in order to study regulation and function of genes and proteins. The biggest hurdle that gene delivery technologies have to overcome is the cell membrane, which is impermeable to negatively charged macromolecules such as DNA and RNA. Numerous different gene delivery methods using either chemical, physical or biological pathways have been used in recent years and are constantly being improved upon. The advantage of recombinant Adenovirus lies in its potential to bind and efficiently enter mammalian cells through its naturally occurring receptor (CAR) (see figure 2). Recombinant Adenovirus is largely used in gene therapy which aims at treating both genetic (e.g. cancer, haemophilia) and infectious diseases (e.g. AIDS) by introducing new genetic material into selected cells. Recombinant Adenovirus can also be used in vaccination by expressing a gene product that triggers an immune response. Recombinant Adenovirus technology is also used to overexpress proteins of interest and to subsequently study their functions. In contrast to prokaryotic or insect-based systems, the use of human cells permits the complex post-translational protein modifications required to ensure the proper folding and post-translational modifications of the protein. In addition, the CMV5 promoter, being substantially stronger than the standard CMV promoter, allows high expression of the protein of interest. Advantages of using Recombinant Adenovirus There are many advantages in using an Adenovirus to introduce genetic material into host cells. Recombinant Adenovirus:
Figure 1:
Figure 2: All these advantages, and the extensive knowledge of viral genetics, have made recombinant Adenovirus the vector of choice for functional genomics research, protein-over-expression, pre-clinical studies and clinical trials. See also our new Adenovirus website: www.adenovirus.com Qbiogene Adenoviral Vector Systems Four different Adenoviral expression systems are offered for the production of recombinant adenovirus. Two of these systems are based on homologous recombination in E. coli, one uses homologous recombination in human QBI-HEK 293A cells, and our most recent system takes advantage of site-specific transposition in E.coli. The major differences between the four systems lie in the mode of recombination and the transfer vectors. With each of the kits, several transfer vectors are available. Some transfer vectors come with a promoter and a poly A site, others benefit from a user-supplied expression cassette. Several of our transfer vectors are available with an MCS (multiple cloning site), whereas others have a single cloning site. In addition, the strength of the promoter, and therefore the level of expression of the protein, is an important consideration for the researcher in the choice of the kit to use. The plasmids, that carry part of the Ad5 genome and are used in the recombination step, are all of the first generation, i.e. ΔE1/E3. The gene of interest, or the expression cassette, is inserted into the deleted E1 region. In addition, a new second generation Adenovirus deleted for the essential protease (PS) gene has been developed by Qbiogene. In the absence of E1, the PS deleted recombinant Adenovirus is completely replication deficient, whereas in the presence of E1, a single round of replication occurs, allowing a greatly enhanced transgene expression without viral shedding which is of particular interest in vaccination, in situ therapy for tumors, protein production, and Adeno-Associated Virus (AAV) production. Furthermore, the ectopic expression of the PS gene permits the positive selection of recombinant Adenovirus (rAD) with 100% efficiency allowing the construction of Ad-based libraries (AdLibTM) having numerous applications in functional genomics studies. The generation of a rAd using the PS deleted viral backbone, as well as the generation of Ad-based libraries, are available as Custom Adenovirus Service. Each kit is available with the principal components and controls for the generation of 5 recombinant viruses and includes a comprehensive applications manual. |
The following table shows the two major characteristics of each system:
| System | Recombination | Plaque Purification |
| AdenoVator™ | Homologous recombination in E. coli BJ5183 cells | Not required |
| AdEasy™ | Homologous recombination in E. coli BJ5183 cells | Not required |
| Transpose-Ad™ | Site-specific transposition of the transposon Tn7 to its specific attachment site attTn7 in HighQ-1 Transpose-Ad™ 294 cells | Not required |
| Adeno-Quest™ | In vivo homologous recombination in human QBI-HEK 293A cells | Required |
Biosafety Notice
CDC (Center for Disease Control) and NIH (National Institute of Health, Office of Biosafety) have defined four levels of biosafety based on a combination of laboratory practices and techniques, safety equipment and laboratory facilities. Class IV provides the greatest overall protection. The components included in our Adenoviral Expression System kits are classified Class II agents.
High-titer Lentivirus Production for in vivo neural labeling NOTE: This protocol describes how to make high-titer lentivirus appropriate for use in vivo in the mouse, rat, or monkey brain, as well as other species (e.g., zebrafinch). The virus works well in cortex, striatum, and many other brain regions, yielding very high infectivity levels (~90%-95% or greater, in mouse brain). With a glial promoter, it also works on glia. It was compiled by Xue Han, Xiaofeng Qian, Mingjie Li, Patrick Stern, and Ed Boyden, as an expanded version of what is found in the paper:
Han, X., Qian, X., Bernstein, J.G., Zhou, H.-H., Talei Franzesi, G., Stern, P., Bronson, R.T., Graybiel, A.M., Desimone, R., and Boyden, E.S. (2009) Millisecond-Timescale Optical Control of Neural Dynamics in the Nonhuman Primate Brain, Neuron 62(2): 191-198. ‘
It is derived from the original protocol used in
Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. (2005) Millisecond-timescale, genetically-targeted optical control of neural activity, Nature Neuroscience 8(9):1263-1268.
Please cite these references if you find this information helpful. If you want to cite this white paper, please cite it as:
Synthetic Neurobiology Memo #2 (2009) Lentivirus production for high-titer, cell-specific, in vivo neural labeling. Online.
0. MATERIALS
Cells:
HEK293FT cells (e.g., Invitrogen R700-07)
Solutions:
D10:
500 mL DMEM (e.g., Cellgro 10-017-CV)
50 mL fetal bovine serum (FBS) (e.g., Hyclone SH30071.03)
5 mL Penicillin/streptomycin (e.g., Cellgro 30-002-CI)
5 mL of Sodium Pyruvate (e.g., Lonza 13-115E)
Mix well; sterile filter with 0.22 micron filter flask (e.g., VWR)
Virus production medium (per 500 mL):
500mL Ultraculture (Lonza 12-725F)
5 mL Penicillin/streptomycin (e.g., Cellgro 30-002-CI)
5 mL of Sodium Pyruvate (e.g., Lonza 13-115E)
5 mL of Sodium Butyrate (100x solution, 0.5M, made from powder (e.g., Sigma # 19364))
Mix well; sterile filter with 0.22 micron filter flask (e.g., VWR)
20% Sucrose solution (per 50 ml)
10 g sucrose (e.g., Sigma)
Bring the volume to 50 ml using PBS
Mix well; sterile filter with 0.22 micron filter flask (e.g., VWR)
Other basic supplies include:
Trypsin-EDTA solution (e.g., Cellgro 25-052-CI)
GeneExpressoMax (Excellgen)
Ultracentrifuge tubes (e.g., Beckman 344058, or whatever your ultracentrifuge requires)
T175 plate: BD Falcon 353112 (e.g., VWR)
100 mm dish: BD Falcon 353003 (e.g., VWR)
140 mm dish: BD Falcon 353025 (e.g., VWR)
1. CULTURING HEK CELLS
2. VIRUS PRODUCTION
PREPARE HEK CELLS (Day 0):
1. Take HEK293FT cells from three 90% confluent 15 cm dishes, and plate onto four T175 plates. Add 25 mL of D10 to each flask. (Should be almost 100% confluent on the next day.)
TRANSFECTION OF HEK-T CELLS (Day 1):
2. Perform a Fugene 6 transfection within 24 hours of plating (when cells are almost 100% confluent). You can also buy transfection reagents cheaply from Mirus. The following recipe is for 1 T175 flask. For 4 such flasks, multiply the recipe by 4. Ingredients:
|
DNA mix |
22 ug lentiviral gene carrier (e.g., FCK-ChR2-GFP, http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=15814) 15 ug pDelta 8.74 (helper plasmid; http://www.addgene.org/pgvec1?vectorid=5682&f=v&cmd=showvecinfo) 5 ug pMD2.G (VSVg, coat protein, from http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=12259) 2 ug pAdvantage (Promega) (this is optional: if not using, use 7 ug of VSVg plasmid) |
|
Fugene 6 |
132 ul |
|
DMEM |
Enough to bring up the total volume to 4.5 mL |
a. Place the Fugene into the DMEM without touching the sides of the plastic tube with the pipetter. Mix with light tapping, then let rest for 5 mins at room temperature.
b. During the 5 min rest, mix DNA (carrier DNA + pDelta + VSVg + pAdvantage) in another tube.
c. Add DNA mix to the Fugene+DMEM mix while tapping the destination tube lightly, and let the Fugene+DMEM+DNA mixture rest for 20-30 mins at room temperature.
d. During the 20-30 mins, replace the culture medium in 1 T175 flask with 16 mL of fresh D10.
e. Then, add the 4.5 mL of Fugene+DMEM+DNA mix to the T175 flask containing fresh medium. Gently rock the flask to distribute evenly.
CHANGE MEDIUM (Day 2):
3. At 24 hours post-transfection, remove the transfection medium from each flask and replace with 20 mL virus production medium per flask. Handle the plates gently, as the virus production process may cause cells to detach.
COLLECTING VIRUS (Day 3 and Day 4):
4. At 44-48 hours post-transfection, collect virus supernatant from each plate into a 50 ml conical flask and replace with 20 ml fresh virus production medium, if you want to collect more virus (we usually collect a second round, as the cells continue to stay healthy and produce high-quality virus for additional time). Spin the collected supernatant at ~1000 rpm for 5 min in a tabletop centrifuge to pellet cellular debris, then filter the supernatant through a 0.45 um (NOTE: not 0.22 micron!) filter flask, pre-wetted with a small amount of virus production medium to reduce protein binding. You can use a 0.45 um syringe filter for small volumes of virus. With this filtered supernatant, proceed to step 6 for this batch, immediately, for best results; storing this filtered supernatant at 4oC for processing along with the second round of virus collection may result in lower effective titers for the virus obtained during the first round.
5. OPTIONAL: If you added a second round of virus production medium in step 4, collect the second batch of virus supernatant at 68-72 hours post-transfection, repeating the process described in step 4, and proceeding to step 6 when you have acquired it. A third round of virus harvesting is usually not warranted for a given set of HEK cells.
6. To ultracentrifuge your coarsely-filtered viral supernatant, to concentrate the virus: transfer the 20 ml of supernatant from each conical flask to ultracentrifuge tubes (sprayed with ethanol in a biosafety cabinet to clean, then air dried). Gently pipette 2 ml of 20% sucrose+PBS solution to the bottom of the supernatant, to make a sucrose cushion, so that light debris will not be collected at the bottom of the ultracentrifuge tube, whereas virus will pellet out. Make sure that each of the tubes (usually 6; if you’ve been following the above instructions for 4 flasks, you may need two “dummy” tubes to balance the 6-tube rotor) is well balanced to avoid ultracentrifuge malfunction: you should weigh the tubes just before centrifuging, to insure balance, to the balance criterion of your ultracentrifuge – often a fraction of a gram. Also, make sure that each of the tubes is decently full – you may want to increase the volumes of virus produced above, or to augment the volumes with sterile PBS at this point, to make sure your ultracentrifuge tubes do not collapse. Spin in an SW-28 rotor in a pre-chilled Beckman ultracentrifuge at 22,000 rpm at 4oC for 2 hours. Follow all manufacturer instructions closely.
7. Carry the ultracentrifuge tubes gently at all times to prevent spillage, but especially now that your virus has pelleted out. Aspirate supernatant very gently, leaving behind the pellet. Observe the pellet – it may appear to be a thin translucent disc, or a white coating, or even be invisible – at the bottom of the centrifuge tube. Put the centrifuge tube upside down on a kimwipe to dry sides of tubes and use a Pasteur pipet to remove any additional medium on the side of the tubes, to prevent the virus production medium from ending up in your resuspension.
8. Resuspend the pelleted virus in a total of 100ul cold PBS (assuming you’ve been following the instructions for four T175 flasks). Add 25 ul cold PBS to each of the 4 centrifuge tubes. Let the PBS sit on the pellet for some time, typically one-two hours at 4oC. Then gently pipette the PBS up and down in each tube, avoiding bubble production, and combine the four resuspensions.
9. Aliquot 2.5 ul-5 ul/tube, or as needed. (We tend to inject on the order of 1 microliter per injection, so aliquotting enough for an entire surgery session is good, to minimize freeze-thaw cycles.) Freeze at -80oC for up to 1 year. During freezing, it is important to use a mammalian cell-freezing box (i.e., which lets temperature drop at ~1oC per minute), for optimal titer preservation.
10. When using: after thawing the virus on ice, centrifuge at 5,000 rpm for 5 minutes, in a refrigerated centrifuge, to pellet out any clumps that may have formed. Keep the virus cold in preparation for surgery (it will last for several hours at 4oC ).
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