Knock-Down Protocol

Transduction is the process by which the genetic material contained in the virus is transferred to the recipient cell. The following protocol provides guidance to facilitate this procedure. It should serve as a guide for experimental design but please be aware that additional optimization may be required depending upon the cell line being utilized. The technical hints at the end of this section lists a number of options that may be implemented in an effort to improve transduction efficiency.

Initial Transduction Test

Prior to beginning any gene knockdown experiments, the transduction efficiency of a cell line needs to be determined. Although most cell types are infected by AdenoSilence library viruses that contain the C01 capsid fiber variant, an initial experiment should be performed to identify the extent of transduction and the optimal Multiplicity Of Infection (MOI; it is the number of viral particles per cell in a reaction). To complete this analysis simply infect the cells with the EGFP-v1 control virus (GAL00104-v1) using a range of MOIs (e.g. 50, 250, 500, 1000, 2500; see below for details on setting up experiments using MOI). The transduction efficiency of each reaction is then determined using fluorescence microscopy or FACS analysis and is typically done three days after infection.

Note: the cells should be observed every day after infection to check for toxicity and/or the start of reporter expression). If the cells exhibit an acceptable level of transduction (generally 70% or more of the cells infected) then choose the MOI with the highest infectivity and lowest toxicity and proceed to use that MOI in subsequent gene silencing experiments. However, if the transduction efficiency does not meet expectations then additional optimization assays will need to be performed. Please note that every new batch of virus should be assayed for the optimal MOI since variability is often observed between lots.

Cell Plating

The size of the reaction plate that is utilized in testing is based upon the objective of the experiment and is user determined. For the initial testing of transduction efficiency it is recommended that experiments are carried out in 96-well plates. The protocol given will be based upon this format. If different reaction sizes are needed simply scale the numbers up or down as necessary.

Protocol for Cell Plating

Seed a 96-well plate the day before transduction using an actively growing culture of cells. The cells should be plated in their normal growth medium. It is perfectly acceptable for the growth medium to contain antibiotics and/or non-human serum since these do not interfere with transduction.
Incubate the plate of cells overnight using the standard culturing conditions for that cell type (i.e., temperature and CO2 concentration).
Ideally the cell density the next day should be about 60% confluent. For example, we typically plate 3000 HeLa cells per well of a 96 well plate (at a total volume of 100 µL).

Transduction and Multiplicity of Infection (MOI)

To set up a transduction reaction some simple math calculations will need to be performed. To accomplish this, a number of variables in the experiment will need to be defined including the cell number, MOI, total number of viral particles needed per reaction, and the titer of virus being tested.

Cell Number—The cell number is just the number of cells present in a well that will be infected with virus. For consistency simply use the number of cells plated the day before transduction. Thus, from the plating example above it would be 3000 cells.
MOI — The MOI is the number of viral particles per cell that is desired during the infection reaction. The optimal MOI may have been determined for a particular cell line already and if so just utilize that number for all AdenoSilence viruses. If not, then a range of MOIs (e.g. 50, 250, 500, 1000, and 2500) will need to be assayed in an attempt to identify the optimal infection amount. The optimal MOI should display a high transduction efficiency with low cellular toxicity. Most MOIs will fall in the range of 100–2500 but they can be higher or lower.
Viral Particles Needed— The total number of viral particles needed per reaction is determined by multiplying the MOI times the number of cells plated per well. For example, if 3000 cells were plated and an MOI of 1000 was desired then the calculation would be:

Titer—The amount of AdenoSilence virus added to a reaction to obtain the total viral particles needed is derived from the actual titer of that virus. Each AdenoSilence virus has a unique viral titer which is listed as Viral Particles per Unit (VPU) on the certificate of analysis (COA) provided for each virus. The VPU is also the same as viral particles per milliliter. To calculate the amount of virus to add to the reaction simply take the viral particles needed (as determined in the step above) and divide it by the titer of the virus. For example, if the titer of the virus from the COA was 1x109 VPU then a continuation of the calculation from above would give:

For consistency the virus that is delivered to the cells is normally diluted with cell culture medium prior to addition. It is recommended that a standard delivery volume of 50 µL be utilized. To determine how much medium should be mixed with the virus simply subtract the volume of virus needed (from the above step) from the total delivery volume. Thus:

Finally, the volumes determined above for virus and medium are multiplied by the number of reaction wells to be tested plus an overage factor is added to account for pipetting errors (1.1X per reaction well). Hence, for a reaction being done in duplicate wells the dilution of the virus would be as follows:
After gently mixing the medium and virus, quick spin the sample, and then add 50 µL per well. Gently rock the plate for 10 seconds to mix the sample. The final volume in a single well of a 96-well plate would be 150 µL.
The cells can then be incubated at the standard cell culture conditions until the desired harvest time. Typically there is no reason to remove the virus-containing medium from the cells following transduction. If viability is a concern the medium can be replaced with virus-free medium at any point 6 hours after the addition of virus.
The above calculations must be carried out for every virus being tested since the titer (or VPU) for each is likely unique. Additionally, if the MOI or cell number changes then the calculations will need to be reworked as well.

Assay Gene Silencing

The optimal time to assay for gene silencing is cell type specific but is approximately six days post transduction. This is the amount of time needed for the adenovirus to efficiently transduce the cells and then initiate shRNA expression. The shRNA then must be processed by the RNAi machinery before facilitating the degradation of the target mRNA. If necessary, a time course experiment with a particular cell line can be set up to identify the best day to harvest for optimal gene silencing.

Validation that gene knockdown is mediated by sequence specific RNAi is critical. The AdenoSilence library contains three viral constructs targeting each gene in the human druggable genome. Testing of these three constructs in parallel in an experiment may help substantiate the legitimacy of the results obtained. For example, an effect caused by any one of the viruses is strong evidence for sequence specific gene-silencing but in rare instances could still be due to non-specific causes (such as “off-target” affects or the interferon response). However, strong evidence is provided if two or more of the viruses targeting a gene show equivalent results. Therefore we recommend that whenever possible, knockdown testing should be assayed using all three adenoviral constructs targeting a particular gene.

Each AdenoSilence library kit is guaranteed to reduce target mRNA levels, which often results in corresponding decreases in target protein amounts and/or loss of function effects. Unfortunately, due to the variability in the stability, expression, and turnover of different proteins, no guarantee can be provided for any changes in protein concentration or function.

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