Transcript for:
Understanding Gene Expression Techniques

Let's begin this lecture with a question. True or false, cells in the eye, let's say the light sensing rods and cones for example, contain different genes than cells in the skin like keratinocytes. If you need some time to think about it, go ahead and hit pause now. A lot of non-biologists would probably answer true to this question, but I hope you knew the correct answer is false.

With just a very few exceptions, all of our somatic or non-reproductive cells are in fact genetically identical. And this is referred to as genomic equivalence. Each somatic cell contains the same genetic material.

Now, of course, this raises an obvious question. If different cell types, neurons and fibroblasts, for example, all contain the same set of genes, then how is it that they acquire their unique sizes and shapes and functions? Well, the answer to that is differential gene expression. What this means is that different cell types express different sets of genes, and that's what accounts for differences between those cell types. not differences in which genes are present.

Now that's a key concept in developmental biology, and so we'll spend a lot of time later in the course talking about how gene expression is regulated. But what I want to do for the rest of this video is tell you about some of the key experimental approaches that biologists use to analyze gene expression. So how can biologists determine when and where genes are expressed during the course of development?

To answer that, we first need to quickly review the basics of gene expression, what Francis Crick referred to as the central dogma of molecular biology. I'm going to assume this is already familiar, so we're just going to review it very quickly here. Now, of course, genes are in the form of DNA, but the DNA really just functions to store information.

And in order for genes to exert some kind of biological function, they have to be expressed. That process begins with... transcription where RNApol2 transcribes an RNA copy of the gene.

Now in eukaryotes, which we'll be focusing on in this course, many newly transcribed RNAs contain introns. The introns are designated here in red, and these will get removed via splicing to join the exons, which are shown here in blue. Now some genes are what we call non-coding, and that means that their RNAs have some function on their own. So, in that case, we're basically done at this step. You'll see some really interesting examples of non-coding RNAs in some future lectures.

But most of the genes we're going to be covering in this course code for proteins. And in that case, the spliced RNA or messenger RNA is exported from the nucleus and then translated by the ribosome in the cytoplasm to generate a protein. And the protein, of course, is what actually does something to control development. Okay, now I want to return to the focus of this lecture.

How can we determine when and or where genes are expressed during development? Well, given what I just reviewed, you can probably already see a couple of obvious possibilities. First, we could try and detect expression of mRNA for our given gene of interest.

And second, assuming we're dealing with a protein coding gene, we could try and detect expression of our protein of interest. There's also a third possibility. which you've already encountered in some previous lectures, called transgenics, which is where we modify our gene of interest so that it encodes a visible protein, like green fluorescent protein. And that lets you track the gene product in vivo or in living organisms, like these fruit flies expressing GFP in their eyes. Okay, so let's take a quick look at specific experimental approaches.

in each of these general categories. And then we'll go into greater detail on them in the next few lectures. And we'll start with techniques for analyzing mRNA expression. And the first one that I want to cover is called in situ hybridization.

This is a classic technique in developmental biology, and it's used to determine when and or where mRNA for a gene of interest is expressed in the embryo. So traditionally, this is done with what's referred to. as a colorimetric detection method. And that generates a deep blue-purple color wherever your gene is expressed.

Like in this example showing an in situ for a gene expressed in the somites and the developing limbs of the chick embryo. Alternatively, you can use a fluorescent detection method and this is called fluorescent in situ hybridization, more commonly abbreviated as FISH. So here's a really nice example of FISH for a gene expressed in the central nervous system of an adult planarian.

So in situ hybridization will reveal the expression pattern for a particular messenger RNA of interest. But sometimes there are situations where you want to obtain a snapshot of the expression levels for many different genes, perhaps all the genes in the genome, either at a particular stage of development or in a particular tissue. And there are a couple of widely used approaches for doing that. The first of those techniques is called microarray analysis.

So microarrays are glass slides that are printed with DNA spots. And the principle of complementary base pairing can be exploited to allow you to hybridize fluorescently labeled nucleic acids in order to detect the levels of mRNA for the gene that's printed in that spot. If that sounds a little bit confusing, don't worry because we'll go into more detail in another video.

For now, just realize that this is a really important approach for determining gene expression levels on a genome-wide basis. There's also another way to do this called RNA sequencing, or RNA-seq for short. And this is a similar approach to microarray analysis, in that it allows you to determine the expression levels for all genes in the genome at once. In this case, the technology used is high-throughput sequencing. And the basic idea is that the more times you obtain sequences corresponding to a particular mRNA in a given sample, the more highly that gene is expressed.

Again, we'll leave the details for another video. So those are some of the key techniques that developmental biologists use to analyze gene expression at the messenger RNA level. But now I want to turn to approaches for analyzing gene expression at the protein level. And the first one in that category is called immunostaining. The prefix here refers to the use of antibodies, which of course are key components of the immune system.

So, antibodies are molecules that bind with high affinity to some kind of target molecule, which is called an antigen. The antigen could be a molecule on the surface of a bacteria cell or a virus or something like that, but you can also generate antibodies that bind to a protein that you're interested in studying. And then if you connect the antibody to, for example, a fluorescent molecule, you have a really great reagent for detecting when and where your protein of interest is expressed.

Here's a great example of what this looks like. This is a zebrafish embryo that's been immunostained with antibodies that detect two different proteins expressed in the nervous system. There are also approaches analogous to microarrays and RNA-seq that allow you to analyze the expression of many different proteins at the same time.

And these are called proteomics techniques. They're a little more challenging than the genome-wide RNA analyses and so they're not quite as widely used in developmental biology, but I did want to mention them. Finally, we have transgenics. And transgenics entails creating a genetically modified organism.

So let's say that you've discovered a new fly gene and you want to determine when and where that gene is expressed. Well there are a couple of approaches you could take using transgenics to figure that out. First, you could just replace your gene of interest with a reporter gene, which is a gene encoding some kind of easily detectable protein, something like GFP.

Alternatively, you could insert the reporter gene just upstream or downstream of your gene of interest, so that when it's transcribed and translated, you get a fusion protein, which joins your protein of interest to the reporter protein. You can sometimes get artifacts with transgenics, so you do have to be aware of that possibility. But the great thing about this approach is that in contrast to the other techniques that I've discussed, which require you to kill the embryo or animal you're analyzing, many transgenic techniques enable you to follow the expression of your gene of interest in live embryos or animals. For example, here's a C.

elegans embryo expressing GFP-tagged versions of histone and tubulin proteins, which lets you follow the dynamics of the mitotic spindle during the first cell division. I should point out here that researchers have created a huge number of different types of transgenic plants and animals. Scientists have even succeeded in generating GFP pigs.

So who knows, maybe you really could have green eggs and ham. Anyway, the take home message here is that changes in gene expression are really, really critical for generating highly specialized cells and tissues during development. And so being able to track those changes in model organisms using the kind of approaches that I've described here is really, really important. And with that, let me encourage you to move on to the next few videos for further information on how all these techniques work.