Explainer: Why scientists sometimes ‘knock out’ genes | Science News for Students

Explainer: Why scientists sometimes ‘knock out’ genes

This technique can help researchers probe the role of a single gene
Nov 6, 2017 — 6:40 am EST
DNA forceps

Scientists don’t really use a forceps when tweaking our DNA. But they do have techniques that allow them to snip out (or turn off) genes in an organism. Afterward, they can investigate the gene’s function by noting what happens when it’s lost.

Natali_Mis/iStockphoto

Each human cell contains some 20,500 different genes. These are small bits of DNA that instruct a cell on what it should do. How do scientists uncover a particular gene’s instructions? One way is to observe how a cell (or a whole organism) behaves when that single gene is absent, or at least not working. Scientists can do that by disabling — or knocking out — a desired gene.

No boxing gloves are required. All they need to start is a single cell. That cell can be a bacterium or yeast. Or it can come from an embryo — the very earliest stages of a developing animal or plant.

The goal of each gene is to make a protein. Proteins are molecules made of long chains. They are the building blocks of most things in our bodies — from bones to muscles to brain. Our bodies make more proteins all the time. But to build them, they have to go back and read the instruction manual — the DNA. Each gene in our DNA produces a different protein.

Often, however, scientists don’t know which gene produces which protein. To find out, scientists can start by knocking out the gene they are interested in. Watching for which protein — or which protein effect — is now gone points to the role of the protein’s maker, or gene.

To knock out the gene, scientists first find or create a version of the gene that doesn’t work. These often will be genes with some serious mistake, or mutation. That mistake makes the gene a total dud — it can’t possibly help produce a protein. They insert this unworking gene into the DNA of a healthy cell. Often, scientists will first attach a “tag” to the dud. Called a reporter gene, that tag can alert scientists when the new gene has been successfully accepted by the cell. How? Reporter genes often produce proteins that glow in green or blue. This lets scientists scout for cells with a tell-tale glow.

How the dud gene becomes part of a new cell

DNA is composed of long strands of genes. It also has sequences of genetic material that play other roles. But sometimes these long strands of DNA become damaged or break. Luckily, machinery within our cells can fix it. Scientists can use that cellular toolbox to slip their knockout gene into the DNA.

The DNA surrounding the gene the scientists want to replace can act like a disguise and helps sneak the dud gene in. When scientists insert their dud gene into a cell, they make sure it has perfect copies of the DNA surrounding the gene that they want to replace. This DNA-costume can fool the DNA-repair system strands. When that system sees the guide DNA, it gets tricked into swapping the dud gene in for the old healthy gene. This “knocks out” the original gene.

Sometimes scientists do not need to perform a full swap. Or they may not know precisely where they need to place the new DNA segment. In these cases, they can insert the reporter gene randomly into the DNA they want to get rid of. Instead of swapping out the old gene, the reporter gene simply gets wedged into the middle of it. When the cell goes to read the gene, the new piece of DNA crammed inside means that the original gene’s instructions no longer makes any sense to the cell. The cell can no longer use this gene to make a protein.

Once a cell is now missing some selected gene, scientists watch what happens. If the cell is a bacterium or yeast, scientists will observe whether it works differently. If the cell is in an embryo, scientists can see how the animal or plant that grows from it differs from normal.

When or where to knock out a gene

Some genes must be working before an embryo can grow into an adult. If scientists want to study this gene, they likely will have to wait until the animal is an adult before they knock out such a gene. Or they may want to see what happens when a gene stops functioning in just one part of the body. Or at some specific time as the cell or animal grows. In these cases, scientists may create something called a conditional knockout.

lab mouse
Scientists can disable, or “knock out,” a single gene in an organism like this mouse. Such animals and other organisms allow them to see how a turned off gene might alter their biology, health or behavior. 
unoL/iStockphoto

To make a conditional knockout mouse (for example), scientists start with the single cell and insert short portions of DNA on either side of the gene they want to knock out. At first, those added DNA sequences don’t change anything about how the cell operates. That means the cell can develop into an adult mouse.

When grown, this mouse now can mate with another adult that has an inserted gene for a protein called Cre recombinase (CREE Ree-KOM-bih-nase), or CR. This gene comes from a virus that usually infects bacteria. The offspring of the two mutant animals will get both added genes, one from each parent. In the pups’ cells, the CR protein recognizes the short DNA sequences surrounding the targeted gene. Then the CR protein can turn that gene on, turn it off, or switch it out for another gene entirely.

But the CR protein doesn’t automatically go to work. In fact, scientists don’t want it to. They want to select precisely when CR turns on.

The gene that makes CR is controlled by yet another gene, a promoter. Something must trigger the promoter in order for a cell to make the CR protein. Usually, some specific drug serves as the trigger. Scientists sometimes use tamoxifen — a drug used to treat breast cancer. Other times they may use an antibiotic drug as the trigger.

Scientists apply the drug to a cell, or give it to an animal in its food. The drug triggers the promotor, and Cre recombinase is made. This protein then knocks out the gene of interest.

Using these various techniques, researchers now can knock out the genes of any organism they choose. There are plants, bacteria and mice with knocked out genes. Scientists can even knock out the genes of human cells growing in a lab.

Power Words

(for more about Power Words, click here)

antibiotic     A germ-killing substance, usually prescribed as a medicine (or sometimes as a feed additive to promote the growth of livestock). It does not work against viruses.

bacteria     ( singular: bacterium ) Single-celled organisms. These dwell nearly everywhere on Earth, from the bottom of the sea to inside other living organisms (such as plants and animals).

bacterium     (pl. bacteria) A single-celled organism. These dwell nearly everywhere on Earth, from the bottom of the sea to inside of plants and animals.

cancer     Any of more than 100 different diseases, each characterized by the rapid, uncontrolled growth of abnormal cells. The development and growth of cancers, also known as malignancies, can lead to tumors, pain and death.

cell     The smallest structural and functional unit of an organism. Typically too small to see with the unaided eye, it consists of a watery fluid surrounded by a membrane or wall. Depending on their size, animals are made of anywhere from thousands to trillions of cells. Most organisms, such as yeasts, molds, bacteria and some algae, are composed of only one cell.

conditional knockout gene    A gene that is inserted into an organism that remains unused until it is triggered by something, such as by giving it some particular drug. The gene is accompanied by a molecule called Cre recombinase, which recognizes short pairs of DNA sequences. The Cre recombinase is controlled by another gene called a promoter. When a drug turns on the promoter, the targeted gene turns on the Cre recombinase. That Cre recombinase then can slice out the knockout gene, stopping it from working. Scientists use this technique to find out what role some gene plays at a particular time in an organism’s life.

Cre recombinase      An enzyme (a type of protein) that is produced by a virus. This chemical can recognize a specific pair of short DNA sequences. When it does, it removes anything between the pair of sequences and now swaps in something new.

DNA     (short for deoxyribonucleic acid) A long, double-stranded and spiral-shaped molecule inside most living cells that carries genetic instructions. It is built on a backbone of phosphorus, oxygen, and carbon atoms. In all living things, from plants and animals to microbes, these instructions tell cells which molecules to make.

embryo     The early stages of a developing organism, consisting only one or a or a few cells. As an adjective, the term would be embryonic — and could be used to refer to the early stages or life of a system or technology.

function     A relationship between two or more variables in which one variable (the dependent one) is exactly determined by the value of the other variables.

gene     (adj. genetic) A segment of DNA that codes, or holds instructions, for a cell’s production of a protein. Offspring inherit genes from their parents. Genes influence how an organism looks and behaves.

genetic     Having to do with chromosomes, DNA and the genes contained within DNA. The field of science dealing with these biological instructions is known as genetics. People who work in this field are geneticists.

infect     To spread a disease from one organism to another. This usually involves introducing some sort of disease-causing germ to an individual.

knock out     (in genetics) To remove or disable a particular gene. The term gets its name because the function of this gene has been knocked out by the procedure. Scientists use this method to find out what the function of a gene might be — by finding out what an organism is like without it.

knockout    (in genetics) The term for an organism that has been bred or engineered in such a way that one of its genes has been disabled, or turned off. The term gets its name from the fact that the function of this gene has been knocked out by the procedure. Scientists can now identify the function of the missing gene by seeing how a cell — or organism — differs when this gene no longer works.

molecule     An electrically neutral group of atoms that represents the smallest possible amount of a chemical compound. Molecules can be made of single types of atoms or of different types. For example, the oxygen in the air is made of two oxygen atoms (O2), but water is made of two hydrogen atoms and one oxygen atom (H2O).

mutation     (v. mutate) Some change that occurs to a gene in an organism’s DNA. Some mutations occur naturally. Others can be triggered by outside factors, such as pollution, radiation, medicines or something in the diet. A gene with this change is referred to as a mutant.

organism     Any living thing, from elephants and plants to bacteria and other types of single-celled life.

promoter   (in genetics) This is a region of DNA that helps another segment of DNA get copied on to RNA. That copying allows the information in that gene to be made into a protein. Promoter regions are located close to a gene that needs their special skills. They contain binding sites onto which special cell machinery will bind. That machinery consists of molecules that copy the DNA onto RNA, so that a protein can be made. In the end, a promoter allows a target gene to produce its protein much more often than normal.

protein     A compound made from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues; they also do the work inside of cells. Among the better-known, stand-alone proteins are the hemoglobin (in blood) and the antibodies (also in blood) that attempt to fight infections. Medicines frequently work by latching onto proteins.

reporter gene  This is a sequence of DNA that scientists attach to another gene that they are interested in studying. The reporter gene is used as a marker. It shows up, like a flag, to mark the activity of some gene of interest. Reporter genes often glow a certain color, or resist a certain drug. This makes it easy to see what the reporter gene — and the gene it’s attached to — is up to.

tag     (in biology) To attach some rugged band or package of instruments onto an animal. Sometimes the tag is used to give each individual a unique identification number. Once attached to the leg, ear or other part of the body of a critter, it can effectively become the animal’s “name.” In some instances, a tag can collect information from the environment around the animal as well. This helps scientists understand both the environment and the animal’s role within it.

tamoxifen     A drug used to treat certain types of breast cancer.

virus     Tiny infectious particles consisting of RNA or DNA surrounded by protein. Viruses can reproduce only by injecting their genetic material into the cells of living creatures. Although scientists frequently refer to viruses as live or dead, in fact no virus is truly alive. It doesn’t eat like animals do, or make its own food the way plants do. It must hijack the cellular machinery of a living cell in order to survive.

yeast     One-celled fungi that can ferment carbohydrates (like sugars), producing carbon dioxide and alcohol. They also play a pivotal role in making many baked products rise.

Citation

Book Chapter:​ R. H. Friedel. “Generating Conditional Knockout Mice.” Methods in Molecular Biology. Volume 693, October 4, 2010, p. 205. doi: 10.1007/978-1-60761-974-1_12.

Website:Knockout mice.” National Human Genome Research Institute. Last updated August 27, 2015.