Four steps to optimizing your shRNA construct
How to pick the best SMARTvector formatting options for your experiment.
Performing RNAi with a shRNA vector is often a straightforward and well documented approach to gene knockdown. But how do you go about finding the correct product and format for your exact experiment? This can be a bit tricky and often a source of frustration, especially if you are new to the field and you find out there are many options. For the Dharmacon SMARTvector lentiviral shRNA platform we’ve got 29 possible formats as both glycerol stock and lentiviral particles! That’s a lot of options. So how do you choose the right configuration for your needs to ensure your experiment goes as smoothly and quickly as possible?
Step one: Constitutive or Inducible promoters
It all starts here, what type of promoter does your experiment require? Do you want to create a stable cell line with constant knockdown, or do you need the temporal control that an inducible promoter can provide? Are you looking to integrate the construct into one cell type and differentiate it into another? These questions can help you determine whether a constitutive or inducible format is most appropriate to your needs. If you require temporal control over when the shRNA is being expressed, and thus the knockdown is occurring, you need to utilize the inducible format (SMARTvector Inducible Lentiviral shRNA). If you want constant expression of the shRNA, then the constitutive promoters are the way to go (SMARTvector Lentiviral shRNA). This is true for both screening and smaller scale applications.
Step two: Glycerol stocks or lentiviral particles
Now we have to ask the second question, what format is best for your needs? Are your cells easily transfectable? Do you plan to package and purify your own lentiviral particles? If so, a glycerol stock might be the right answer. However, what if your cells are difficult to transfect or you need to control the level of particle integration (e.g. for pooled screening)? This is where lentiviral particle formats are most useful. While you can always purchase the glycerol stock format and package on your own, that can be a daunting challenge when you think about the time to package, collect, purify, and titer the lentiviral particles. This is where just going right to particles is helpful, sure, it may cost a little more upfront, but how much time and effort are you saving that you could be dedicating to other requirements in the lab? Lastly, are you looking to generate a stable cell line with the shRNA integrated in the cells genome? Lentiviral particles are the perfect platform for this approach. At the end of the day, the good news is there is no real right or wrong answer to this question; select the format that is most applicable to your experiment, your budget, and to your comfort level.
Step three: Selecting promoter options
This can be where the selection process can get to be a little tricky. The SMARTvector Lentiviral shRNA platform is available in four (4) inducible promoter options and the constitutive format has seven (7) choices! So how can you figure out the best one for your cells? The best method is the old scientific standard, empirical testing. Test the options to determine which promoter is the best candidate for your cells of interest. There are two great tools to help with that: 1) the SMARTchoice Promoter Selection Plate, and 2) the SMARTchoice Inducible Non-targeting Control 4-Pack. These tools allow you to quickly and easily perform a functional assay to determine which promoter is the ideal candidate for your cells; essentially all things being equal, you’re looking for the most green cells under a fluorescent microscope or by using FACS. While we’re on the subject, let’s address one small potential (and common) misconception. You may have heard that for human cells you have to use a human promoter and for mouse cells you have to use a mouse promoter; this is not entirely true. Promoters are tools, they drive the expression of whatever is downstream, some work better in one cell than others; but they are just tools and there is no species specific requirements, in fact, here’s a picture:
What you can see is that in the A549 cells (a human line) the murine CMV (mCMV) promoter is the ideal candidate for the job, sure human CMV (hCMV) works, but the mouse works better. At the end of the day it’s about getting the right tool for the job. What’s also true is that the stronger the promoter activity in the cell, the more of the shRNA is going to be expressed; that means stronger knockdown and an easier to decipher experimental result. This is why you want to empirically test and determine the best promoter for your cells; you give yourself the absolute best chance of experimental success.
Step four: Reporter options
Arguably this is the easiest step in the entire process; the SMARTvector inducible platform is available with your choice of either a green or red reporter and the constitutive vectors are available with a green, red, or a no-reporter option. You can use the reporter to select transduced/transfected cells from the background and to easily optimize your delivery conditions. Choosing the right one is a simple matter of looking at what channels are already in use, or are likely to be in use, in your experiment. We find most people default to green, but if you think you might need green elsewhere later the red is a beautiful choice. If you have both red and green tied up, or you are working in a system where any extra fluorescence may cause a challenge with the experiment, the no-reporter option is your best bet. Remember, you can always use puromycin selection to generate a stable population.