Some Thoughts On Creating Stable Cell-Lines

Creating stable cell-lines can be a straightforward or tricky process. In this post I will share some techniques or quirks, which I found worked when creating stable cell-lines.

* Use cells with low passage number – Thaw out your cell stocks^1,2 and start with “new” cells with low passage number.

* Culture cells in antibiotic-free media – By cutting out penicillin/streptomycin, I found increases in my transfection efficiencies. It also avoids any possibility of the penicillin/streptomycin interfering with the selection antibiotic during the selection process.

* Make the selection media fresh – Have aliquots of your selection antibiotic at a higher concentration and dilute it into fresh media each time you need to do a media change.

* Change the selection media daily – After transfection, change the selection media daily up until you reach the limiting dilution stage. It may seem excessive or wasteful but I have found that by having freshly made selection media and a daily media change ensures that the untransfected cells are effectively killed off.

* Have high concentrations of your antibiotics aliquoted – Freeze-thaws and heat-cool cycles can affect the efficacy of some antibiotics so to avoid any issues calculate the approximate amount that you will need for selection and aliquot small amounts into separate tubes.

Cloning Tip – How Not To Expose Inserts To UV During Preparation

As mentioned in a previous post¹ there is a way that you can isolate PCR-amplified inserts from DNA agarose gels without exposure of the insert to UV.

First, create replicates of your insert by amplifying multiple PCR reactions.  I generally prepare between 3-5 replicate reactions to increase my insert yield. Prepare a 1% agarose gel + ethidium bromide. After your PCR goes to completion, cool the reactions to 4 degrees or leave it on ice. Add loading dye and load the reactions onto your gel. In the lanes indicated with the letter “L”, add your DNA ladder. I have used a 100bp ladder as an example.

Run the gel. Once completed, remove the gel from the tank and cut the gel as indicated by the red dashed line. Take the smaller portion of the gel to a dark room and visualize on an open UV box. Take a blade or scalpel and mark the gel by nicking the gel edge just above and below the band of interest (green) as indicated by the asterisks. 

Reassemble the entire gel at your bench and then using the nicks made to the gel and the ladders as a guide, carefully cut out the area where your replicate bands of interest are likely to lie as indicated by the dashed red lines.

Gel purify the gel cut-outs containing your bands of interest (green) and proceed with your cloning protocol.

Cell Culture and Antibiotics

There are arguments for and against the use of antibiotics (e.g. penicillin/streptomycin) in cell culture. On the one hand, it is a useful prophylactic to prevent cultures from contamination by providing a layer of protection. However, arguments have been made against its use with claims that constant usage allows for unintentional selection of antibiotic resistant bacteria, researchers become reliant on it and becoming lax in applying aseptic techniques and good cell culture practice.

I am inclined towards the latter view of avoiding the use of antibiotics, but mainly because antibiotics can interfere with particular experiments, namely transfections. However, I do find that it is better to use aseptic techniques because it is better to know quickly that your cells are contaminated rather than have low level contamination hang around which is not visible. The danger of low level contamination is that it will affect and alter your cells and interfere with your experiments.

In regards to students or researchers learning cell culture for the first time, it is probably better for them to see how lapse in aseptic practice can easily lead to contamination. People learn from their mistakes and will only improve with practice.

TOPO TA Cloning – Adding 3’A Overhangs

The TOPO TA cloning kits for subcloning offer an easy way to subclone effectively, provided you can get it to work for you.  The topoisomerase I in which the kit relies on requires the presence of 3’A overhangs on the DNA inserts in order to catalyze the reaction joining insert to vector. Ironically, the enzymes and other components required to add the 3’A overhangs are not supplied with the kits and the protocol provided in the instruction manual is not what I consider ideal.

I have personally never followed the 3’A overhang procedure set out in the product manuals provided; instead, I used my own, which I believe works out more efficiently.

The following is a quick and general run-down of how I clone using the TOPO TA subcloning kits. The focus will be on the addition of 3’A overhangs.

Insert Preparation

* Setup PCR reactions to amplify your insert. Use a proofreading DNA polymerase.

* Run your DNA gels and cut out your insert. If you do not want any possibility of point mutations or DNA breakage, try excising your bands without exposure to any UV.

* Gel purify your gel cut-outs. I recommend using a kit such as Qiagen's QIAquick Gel Extraction Kit. Elute/resuspend the DNA in nuclease-free water.

Adding 3’A Overhangs

Proofreading DNA polymerases have 5' to 3' polymerization and exonuclease activity as well as 3’ to 5’ exonuclease activity (proofreading). It is the 3’ to 5’ exonuclease activity of a proofreading DNA polymerase which enables it to remove any base-pair mismatch, including any overhanging bases, thereby generating blunt-end PCR products. In contrast, Taq DNA polymerases lack the 3' to 5' exonuclease activity, so while Taq enzymes are not suitable for generating inserts for cloning, they are useful for TA cloning for the addition of 3’A overhangs.

* You will need a Taq DNA polymerase which does not have 3’ to 5’ exonuclease activity. Check the product information sheet. An example of such a Taq is Thermo Scientific's Red Hot Taq DNA Polymerase.

* Using the Red Hot Taq as an example, set up the following:

For x1 reaction:

10x PCR Buffer à 2.5ul

MgCl₂ à 2ul

dATP (10mM stock) à 0.5ul

Red Hot Taq à 0.1ul

Insert DNA (from gel extraction) à 19.9ul

Incubate in a PCR thermal cycler à 72 degrees for 30 minutes. Do not cycle. Cool on ice or 4 degrees when complete.

DNA Precipitation

Cool your reaction on ice and proceed to precipitate your DNA inserts.

* Take the above 25ul reaction and add 2.5ul (which is 1/10^th volume) of 3M pH5.2 NaAc (sodium  acetate). Tap or gently vortex to mix.

* Add 62.5ul (which is 2.5 volumes of ice cold absolute ethanol). Tap or gently vortex to mix.

* Incubate the entire reaction on ice for 30 minutes.

* Centrifuge at 16200xg for 20 minutes.

* Aspirate the supernatant and wash the pellet with 500ul of 70% ethanol.

* Centrifuge at 16200xg for 5 minutes.

* Aspirate the supernatant, air dry the pellet and resuspend in 10ul nuclease-free water.

* Use 4ul for TOPO cloning reaction.

Things To Consider When Ordering Published Primer Sequences

When you see a set of primers in the materials and methods section of a journal publication, it is tempting to order the sequences provided. Before doing so, it is good practice to check the primer sequences to make sure that they are suitable for your needs. In other words, make sure the primer sequences published are suitable for your needs in regards to:

* Cell type – some cell types may have little to no expression of your GOI.

* Tissue type – as with cell type, there may be little to no expression of your GOI.

* Species and percent homology if the species are different.

* Whether the region targeted in the transcript will give you the expected product size when PCR products are run on a gel.

* Primer direction – sometimes the primer sequences provided in a journal article may not be in the correct format for ordering. For instance, if a reverse sequence rather than the reverse complement sequence is provided in the publication, you will have serious issues when you start experimenting with the primers.

* Purpose of the primer – it is important to see what experiments the sequences published were actually used for. A primer set used for expression cloning will not be suitable for real-time PCR.

* The primer sequence published – you want to make sure that the sequences provided are accurate so check by blasting the sequence. It is not uncommon for authors to make typos when entering in their sequences during manuscript preparation.  

Data Analysis – T-Test vs Mann Whitney U

For those unfamiliar with statistics, it can often be confusing when deciding which test to apply to analyse data in order to determine whether changes observed are indeed statistically significant (i.e. p<0.05)

The following will provide some guidance on how to analyse data between two groups (e.g. placebo vs drug treatment, normal vs diseased, light vs dark, etc).

What Are The Differences?

The t-test is a test between population means. They are parametric tests and should only be applied to data that is normally distributed. In contrast, the Mann-Whitney U (MWU) test is a test of differences in medians as well as the shape and spread of the data. It is a non-parametric test that can be used as an alternative to the t-test on data that is not normally distributed. However, it should be noted that the MWU test can be applied to normally distributed data.

The number of data points or sample size can also affect the choice in tests. If you have large datasets that have a normal distribution, a t-test can be very powerful. But if you have a small number of data points (e.g. less than 6 data points), a MWU test would be preferable to a t-test since the data is unlikely to have a normal distribution.

What Do I Use?  

To determine which test to apply, you will first need to establish whether your experimental data is normally distributed. For illustrative purposes, a mock dataset (below) will be used. The dataset below is from two groups (normal vs diseased). The sample type is coded and blind to the experimenter.

There are a number of normality tests for you to choose from, but if you have a set of data with less than 2000 data points, as above, try using the Shapiro-Wilk normality test. The null hypothesis is that your data points belong to a normal distribution; reciprocally, your alternative hypothesis is that your data points do not belong to a normal distribution. If p<0.05, your data does not have a normal distribution. For the above dataset, the results of running the Shapiro-Wilk test is as follows:

n = 24

Mean = 66.33333333333333

SD = 21.25142791451429

W = 0.9437512788610762

Threshold (p=0.01) = 0.8840000033378601

Threshold (p=0.05) = 0.9160000085830688

Threshold (p=0.10) = 0.9300000071525574

Here, p>0.05 at all thresholds. Accordingly, the alternative hypothesis is rejected and we conclude that the data points have a normal distribution.

** The results above were calculated using an online Shapiro-Wilk calculator.   

For sample sizes greater than 2000, you can use the Kolmogorov-Smirnov test. If you prefer to visualize the data in graphical form, try using a normal probability plot or quantile-quantile plot.

Other Ways To Test Normality

Aside from the normality tests mentioned above, there are other normality tests available. For their description, please refer to the following link.  

Static On Film

Have you ever gotten strange artifacts on your films that look like this:

This is the result of static electricity and if you have acclimatized yourself for long enough in the darkroom; you can even see when the static electricity sparks go off on the film.

These artifacts are a pain because they can ruin a perfectly good experiment. For something like western blotting, getting an artifact like the above that covers up your result is annoying but most of the time, you can make a few repeat exposures before the ECL substrate gives out on your membrane. However, if you are working with something that takes weeks to months to develop on film (e.g. S³⁵ in situ hybridization), getting an artifact that covers up some of your most important image results can be very devastating as the entire experiment will probably need to be repeated.

I have had the unfortunate experience of having these artifacts crop up in my experiments. Over time, I came to realize that the main problem lied with the lab’s air conditioning.

Static electricity is a build up of electrons (negative charge) within or on the surface of an object that is retained until it can be released by an electric current or electrical discharge. Water is a conductor of electricity, so in a humid environment, the moisture in the air absorbs and can evenly distributes excess charges, thereby allowing excess charge to leave objects.

My Experience

During my time working in a lab housed in an old building, I have never once gotten an artifact like the above on any of my films (and I had done a lot of film work). Fast forward to my new lab, which was brand new, the artifacts appeared almost all the time I did any film work. My technique, equipment, and reagents were pretty much the same so the only real difference was the lab itself. I ultimately concluded that the problems came from the level or moisture in the air. In my old lab, there was no air conditioning or windows that actually opened. As such, the lab was always humid. In contrast, my new lab was fully air conditioned and the air was always very dry. I tested my theory by having the air conditioning turned off in the new darkroom; while it did get pretty hot and humid in there, importantly, the artifacts stopped.

Suggestions

If you are having problems with artifact on your films cause by static electricity:

* You can try using films containing an anti-static layer, although I still found the artifacts cropping up on them.

* Try preventing the static build up beforehand. If you are using transparency film to sandwich your western blotting membranes, leave them in a humid area. The same goes for the autoradiography film or hyperfilms.

* If possible, switch off the air conditioning (at least for the darkroom).

* If possible, use a humidifier.