Site Loader

In this lesson, we’ll learn about the steps to create a knockout mouse. Not to be confused with a mouse and a pair of boxing gloves, a knockout mouse is a model organism that has been genetically manipulated to disrupt a gene’s function so that we can learn about the purpose of that gene.

Genetic Manipulation of Model Organisms

We’re at the point in science where we can figure out the DNA sequence of every living thing. However, our next task is to figure out what all this sequence means. How do we know what each individual gene does?Let’s say we have a gene of unknown function in the human genome. We’ll call our gene UFO. This will stand for ‘unidentified funny-looking order of bases.

Best services for writing your paper according to Trustpilot

Premium Partner
From $18.00 per page
4,8 / 5
4,80
Writers Experience
4,80
Delivery
4,90
Support
4,70
Price
Recommended Service
From $13.90 per page
4,6 / 5
4,70
Writers Experience
4,70
Delivery
4,60
Support
4,60
Price
From $20.00 per page
4,5 / 5
4,80
Writers Experience
4,50
Delivery
4,40
Support
4,10
Price
* All Partners were chosen among 50+ writing services by our Customer Satisfaction Team

‘ There are several ways to try to figure out what this UFO gene does. First, we’d probably look to see what other genes have similar sequences to this one. If these similar genes have a known function, our gene might code for something similar, too.However, if this UFO gene is completely different than anything we’ve ever seen before, this approach might not be much help. So, one way to investigate a function of a new gene is to remove the gene, and then, observe what happens to the organism.Let me give you a little analogy.

This is like taking away a kid’s toy and observing the reaction. Maybe the child won’t care. Maybe the child won’t even notice. Maybe the child will attach itself to another familiar toy or maybe find a new one. Or, maybe the child will start a horrendous temper tantrum. Until you take it away, you just don’t know the value of that toy to that child.

Until we take a gene away from an organism’s genome, we don’t know the value or purpose of that gene to that organism.

A Knockout Mouse

So, how do we take away this UFO gene? We might be interested in how this UFO gene functions in humans. But, we can’t take genes away from humans very easily, or ethically. Therefore, the first thing to do is decide which model organism you’ll use.Remember that a model organism is just any non-human organism used in research to answer a scientific question. We have a lot of choices in model organisms that can be used for this purpose. In this lesson, we’ll talk about a basic method to delete the UFO gene in a mouse.

Mice are popular model organisms because they are mammals, like us, with similarity in genome to humans. They are also reasonably cheap, easy to raise, and have a short generation time.Deleting a gene or disrupting a gene’s function in a mouse creates what’s known as a knockout mouse. We have ways to remove genes in other organisms but it might be called something different.

You can remember this term by thinking of it this way – it’s like we put on a pair of gloves and punched a hole in the mouse genome, effectively ‘knocking out’ that gene’s function.Making a knockout mouse takes a lot of steps. So that you can get a feel for the complexity of creating a knockout mouse, we’ll go through the basic overview of one way this can be done.

However, there are a lot of details that we won’t get into. Also, keep in mind that there is always more than one way to do something. In addition, although this method will be similar in other model organisms, it will not be identical.Tens of thousands of genes have been ‘knocked-out’ in mice, but that still leaves plenty more to be investigated.

Targeting Vector

Step 1 is to create a targeting vector. A vector is just a way of getting something somewhere. A targeting vector, then, is something that is targeted for a specific area.

In this case, it means creating a piece of DNA that can be targeted to the right place in the genome, right at the gene of interest.The best way to understand this is to look at the drawing below. Here, we see a portion of the mouse genome with the UFO ‘target’ gene represented by a box. This target gene is the one we want to knockout. The boxes on either side of the UFO gene represent some of the DNA to the left and right.

To create a targeting vector, you pretty much recreate the DNA on either side of the UFO sequence. Keep in mind that although we are drawing this as a straight line, this targeting vector is usually circular with additional pieces of DNA on it.

Example used to show how to create a targeting vector
example used to create targeting vector

These matching pieces of DNA are known as homologous. Instead of putting UFO in the middle of the targeting vector, you put a reporter gene. The general idea of this strategy will be to replace UFO in the mouse genome with the reporter gene, which will then be expressed instead of the original UFO gene.We can select for this reporter gene.

Therefore, this could also be called a selection marker. For example, let’s say our selection marker or reporter gene coded for bacterial resistance to an antibiotic. Normally, mouse cells do not have this gene and will die in the presence of the antibiotic. However, if you correctly replace UFO in the mouse genome with this antibiotic resistance gene, then the mouse cells would now be resistant to the antibiotic.

You could then use that antibiotic to select for cells that were expressing the reporter gene and select against cells that still had UFO.In addition, there’s another piece of DNA called a negative selection marker. This piece of DNA will code for something that will allow you to select against instances when the reporter gene inserts into the wrong place in the genome – somewhere other than the UFO locus.

Steps in Creating a Knockout

Once we have our targeting vector, step 2 is to insert the targeting vector into mouse embryonic stem cells. Remember that embryonic stem cells, or ES cells, are special non-specified cells in an embryo that can differentiate into any cell type. This concept is key because we are changing the genotype of these stem cells; essentially, we are removing a specific gene from them. When these cells grow, divide and differentiate, all their daughter cells will also be missing our UFO gene. Also, it’s important to note that these ES cells are being grown in a Petri dish.

Once we insert the targeting vector into ES cells, it will undergo homologous recombination with our target UFO gene. Remember that the word ‘homologous’ contains the prefix ‘homo’ meaning the same or similar. The same pieces of DNA will line up and recombine, giving us our reporter gene in place of the UFO gene. This will happen in some cells but not all.Therefore, step 3 is to find ES cells that have the UFO gene knocked out correctly. We can do this by using the reporter gene and negative selection markers from the targeting vector we created in step 1.

Therefore, this is a selection step.Now that we have ES cells with the right genotype, in step 4, we will inject correct ES cells into a mouse embryo. These ES cells that we transplanted are heterozygous for the knock-out. In other words, they will have one wild-type copy of UFO on one chromosome but are missing their second functional copy of UFO.

Additionally, this mouse embryo has both knock-out ES cells and the original non-knock-out ES cells. Therefore, the mouse embryo will grow up with both kinds. Then, we let this little embryo grow up inside a mommy mouse.In order to get a mouse that is homozygous for this knock-out, we’ll take the babies that are born and use them in step 5. Here, we will mate mice to obtain a homozygous mouse.

We will use different molecular methods to confirm that we have obtained our knockout mouse.Now, once we complete these steps, step 6 will be to observe the knockout mouse! We’ll look for all kinds of phenotypic, or physical, changes. The mouse might not live to adulthood, or it might be fine. The mouse might have specific health problems, like weight-gain or heart disease. What we observe will help tell us what the UFO gene does in a mouse!

Lesson Summary

Whew. Now, let’s review today’s lesson.

In this lesson, we created a knockout mouse. A knockout mouse is genetic manipulation where we delete a gene or disrupt a gene’s function in a mouse model organism.This is a complex, multi-step process where we create a targeting vector that essentially will replace a gene that we are interested in studying with a reporter gene. The purpose of this process, however, is to study the function of an unknown gene.

By removing this gene, we can observe the effects on the mouse and gain insight into the gene’s function and importance. This can be done in other model organisms, as well; however, the method varies.

Learning Outcomes

After this lesson, you might be able to:

  • Clarify the importance of creating a knockout mouse
  • Expound upon the function of a targeting vector
  • Refer to the steps necessary for creating a knockout mouse

Post Author: admin

x

Hi!
I'm Eric!

Would you like to get a custom essay? How about receiving a customized one?

Check it out