“Suppressing the function of a gene or inactivating it using gene manipulation methods in a DNA sequence of a gene is called a gene knockout process.”
The gene knockout method is one of the traditional and most trusted methods used since long for studying the function of a gene or a group of function for different genes.
A gene is a functional piece of a DNA which encodes a protein, by inactivating a gene either by removing DNA sequence, altering DNA sequence or introducing a mutation inhibits the normal function of a gene- loss of function.
That gene is then inserted into the germline cells of a model organism and allow to grow. Using artificial vectors it can be inserted into the growing embryo.
If a gene knockout performed well, a visible phenotypic variation can be observed or alteration in the biochemical phenotype can be reported. This is the simple explanation of gene knockout.
In the present article, we will discuss some of the interesting aspects of gene knockout and related topics. Furthermore, we will talk out some of the major differences between gene knockout and gene knockdown at the end of the article.
- Introduction to gene knockout
- Steps in gene knockout
- How to confirm gene knockout
- Methods of gene knockout
- Gene knockout vs gene knockdown
Introduction: Gene knockout
The gene knockout is a molecular genetic technique used to study the function of a gene, abbreviated as KO.
Let’s take an example, suppose we wish to study how mice coat hairs are developed. For let say an MCH gene encodes mice coat hair.
If we wish to study how the MCH gene works we have to suppress its activity or inactivate it. For that, we can use different techniques,
We can introduce a mutation into the DNA sequence of the MCH gene, we can inactivate some of the promoter sequences which regulates its gene expression or we can remove the entire MCH gene.
Then the final version of the (inactive) gene is introduced into the vector and which are inserted into the embryonic stem cells.
The model organism- transgenic mice developed from the homozygous cell types may not have hairs on its coat, we can call it “naked mice”.
The MCH gene might also be linked to some other function as well, that functions are also suppressed in the homozygous mice and can be studied.
The genetically altered mice are called gene knockout mice or gene knockout organism or gene knockouts.
Similarly, the process of knocking out two genes are called double knockout (DKO).
However, along with with the physical examination, several other methods such as polymerase chain reaction or DNA sequencing can also be used for confirming or validating the results of the gene knockout, that we will discuss in the latter part of this article.
The gene knockout is practised in the plant, animal and microorganism studies for studying different functions of an organism.
Definition of gene knockout:
In simple words we can define it as “a process of suppressing gene function by gene manipulation is called gene knockout.”
“Using either gene manipulation or artificial mutagenesis, loss of function of a gene can be caused to study the function of a particular gene in an animal model organism is called gene knockout method.”
Process of gene knockout:
Selecting a gene for knockout:
The very first step in any of the genetic engineering experiment is to select the target, here, the target is a gene which we want to study or whose function we wish to understand.
After that, some of the dry lab work is done in which the structure, length and other parameter related to our gene of interest are studied.
Based on that, the best-suited plasmid for the experiment is selected. But before that, the gene of interest is identified and mapped on a chromosome.
Construction of vector:
Vector is a vehicle used to transfer our gene of interest or any other DNA sequence to our target cells, a plasmid is generally used for it.
The plasmid is the extrachromosomal DNA of a bacteria used for genetic engineering experiments. Read more on plasmid: Plasmid DNA- Structure, Function, Isolation And Applications.
BAC- bacterial artificial chromosome vector is used for gene knockout experiments.
Suppose we have introduced a frameshift mutation into our DNA sequence, which inhibits the protein formation.
We have removed some of the ORFs- open reading frames from the gene and inserted an altered gene into the BAC.
Now for safer side, to validate our results a marker DNA sequence is also introduced in it, generally, an antibiotic resistance gene is used for it.
With it, some of the other important DNA sequences such as the origin of replication, promoter sequence and recognition sequences are inserted into the plasmid as well.
Now our plasmid is ready for the transformation.
“A marker gene is inserted only to make the insert detectable for reporting the results, it works as a reporter.”
NeoR gene- neomycin resistance gene is one of the popular reporter or marker sequence used in the gene knockout studies, in the presence of it mice cells dies (because the NeoR gene is generally not present in mice).
The gene is inserted between the left and right arm of the plasmid or target vector, once the left and right arm recombination with the gene of interest, the NeoR gene inserted into the target nucleic acid.
Sometimes a negative selection marker gene or negative reporter gene is also used along with it.
Insertion into ES cells:
Now using artificial methods such as electroporation, sonication or microinjection, our plasmid is inserted into the ES cells.
Why ES cells:
Embryonic stem cells can be divided faster and divided into any types of cells.
The ES cells have the power to develop into the mature mice tissues.
The electrophoration method is one of the best technique used in gene knockout by scientists in which under the electrical current, a gene is inserted into the cell.
Now our plasmid is inside our target cells i. e ES cells.
If it finds the target gene, recombination will occur and a mutation is inserted into the target gene.
We have also used one marker gene sequence thus along with the mutant gene sequence, our marker gene sequence is inserted into the genome of transformed cells as well.
Now our transformed cells are grown into the neomycin containing media so that the cells containing the NeoR gene can grow. And we can differentiate between NeoR containing cells and cells without the NeoR gene.
Confirming the insert:
When we grow our cells under in vitro conditions, it is possible that some cells may be transformed or some cells may not.
Now using the polymerase chain reaction, the insert can be confirmed. For that, DNA is extracted from the cultured cells.
A set of primers specific to our marker DNA sequence is used for achieving amplification. If the amplicons are observed, cells are transformed otherwise our experiment is failed.
Suppose the ES cells are transformed successfully, we can now call it genetically modified cells.
Injecting into the embryo:
Now pick transformed cells and insert them into the developing embryo of our model organism. Let it grow normally.
Now the embryo of our model organism has two types of the cell population, one wild type and one altered (transformed) cells, this animal is called chimeric.
Our chimeric animal is now genetically modified, in the next step, we breed it with the normal animal which produces offspring of two different genotypes: one with homozygous normal or another animal with homozygous altered genotype (and heterozygous as well).
Now our gene knockout animal is constructed, scientists can examine it for measuring different parameter related to our gene of interest.
Physical, biochemical or genetic parameters related to our gene can be studied. After that using the PCR amplification method, results of gene knockout can be validated.
The entire process of gene knockout is represented in the figure below,
How to confirm gene knockout?
Validating gene knockout is one of the crucial and very important part of the entire experiment. Though many different methods are used to do so, one of the popular methods, nowadays is, polymerase chain reaction.
The polymerase chain reaction is one of the widely used methods and most trusted for most experiments.
Here for confirming or validating gene knockout, two sets of primers are used.
The one set of primers are designed from the flanking regions of the target sequence and another set of primers are designed to the sequence of marker gene i.e. the antibiotic resistance gene.
Now note down one of the important results of the experiment: if the target gene is recombined, the antibiotic gene is transferred to the target genome.
Therefore, if we get the DNA band in the PCR reaction with the primer set complementary to the marker gene, our experiment is successful.
On the other side, once the target gene is removed, the flanking primers specific to the gene of interest can not be amplified and the DNA band can not be obtained.
However, as a control reaction, used the wild type DNA so that a DNA band with primer set 1 can be obtained.
Once the amplification reaction is completed, the results are validated using the agarose gel electrophoresis. The results of PCR can be visualised on 2% agarose gel.
Why knockout mice?
Mice with an inactivated gene of interest created to study the function of that particular gene is called knockout mice. We are using the mice in the genetic engineering studies and knockout studies because of the similarities between the genes of human and mice.
99% of human and mice genes are similar, thus instead of using human embryo directly for the experiment, using mice is a wise decision.
Due to several ethical issues associated with human embryo studies, scientists are using mice for gene knockout and gene knock-in studies.
Methods for gene knockout:
For constructing artificial gene knockout organism several methods are used to silent or remove the gene of interest.
Methods for gene knockout are enlisted here:
- Gene silencing
- Conditional knockout
- Homologous recombination
- Gene enditing
- Knockout by mutation
RNAi or RNA interference is cells natural mechanism used for post-translational modification such are gene silencing or suppressing gene expression.
Smaller double stranded-RNAs such as siRNA or shRNA are used for mRNA-target specific gene silencing.
Using artificial methods or liposome, siRNA or shRNA can be introduced in the cell which is recognised by the cells defence mechanism and processed in the RISC.
The “Ago” endonuclease helps to separate the guided-strand and passenger strand in which the guided strand binds to the target mRNA and inhibits protein formation.
Either the guided-strand binds to exact complementary sequences on the mRNA or on the 3’ untranslated region of the mRNA and prevents gene expression.
We have covered a series of articles on RNA interference. Read it here:
One of the disadvantages of the RNAi is off-target gene silencing.
The conditional knockout method is used to inactivate the gene in a specific tissue at a specific time for a specific function. In addition to this, it is done in the adult animal instead of during the embryonic stage through the process of homologous recombination.
Cancer like lethal conditions can be studied by the conditional gene knockout method using mammalian model organism.
The Cre-LoxP method is adopted for studying the conditional knockout in which the site-specific recombinase “Cre” recombinase the short target sequences called LoxP.
The conditional gene knockout method is widely used to study the effect of different diseases on model organisms, for example, the effect of BRCA1 gene mutation is studied using the knockout mice model.
One of the traditional and widely used methods for studying gene knockout in genetic engineering is homologous recombination.
Exchange of the nucleic acid between identical or homologous sequences occurs through the homologous recombination.
And scientists are using this idea to insert the gene of interest, in place of our target gene. Identical DNA sequences of up to 2Kb are inserted in the vector along with the antibiotic resistance gene and incorporated in the target genome using artificial methods such as electroporation, microinjection or sonication.
Once the vector is inserted into the cell it recombined with the target DNA sequence, our DNA of interest with the antibiotic resistance gene inserted into the target genome.
This method is one of the simplest and effective methods used since long, however, the efficiency is very low. See the image,
Gene editing is one of the emerging tools in recent day genetics where scientists are using nucleases to remove nucleic acid using homologous recombination.
ZFNs, TALENs and CRISPR-CS9 are gene-editing nucleases used in genetic engineering.
ZFN and TALEN are the traditional and outdated method, although the CRISPR-CAS9 is more effective and efficient.
CRISPR- clustered regularly interspersed short palindromic repeats found naturally in bacteria, using CAS9 protein- nuclease it cuts the exogenous nucleic acid to protect the bacteria.
Using this mechanism in gene therapy, scientists are able to cut and insert new DNA at the location they wish to study.
All three methods are uses site-specific nuclease action to introduce a double-stranded cut in a DNA which is repaired by non-homologous end-joining by cell’s own DNA repair mechanism.
A frameshift or deletion mutation can be induced in a DNA sequence utilising this process which results in the nonfunctional protein product.
Well, the entire concept is to remove or edit gene; if you want to know more on gene editing and CRISPR CAS9 please read our previous article on it: What is gene editing and CRISPR-CAS9?
Gene knockout by mutation:
A loss of function mutation can help to suppress gene function by creating a mutation. Either using chemical mutagens or physical agents, scientists can introduce mutation in a gene, however, the specificity and accuracy are very low.
Other methods as discussed above; nuclease mediated cleavage or using the synthetic dsRNA molecules mutations can be incorporated into the DNA sequence.
Remember, in the gene knockout method, our prime goal is to make a gene nonfunctional.
Anyway coming to another important point of this topic, what is the difference between gene knockout and gene knockdown?
Well, at first instance, both the term looks similar, although both are different techniques and used for a different purpose.
Gene knockout Vs gene knockdown
Making a gene non-functional is called gene knockout, as we are discussed in the entire article while reducing the expression of a gene is known as gene knockdown.
In a simple language, we can say in the gene knockout method we are making a gene inactive totally, while in the gene knockdown, the gene is active but the expression of a gene is reduced to know its activity in a particular cell type.
One of the best methods for inactivating a gene is by introducing a mutation- gene knockout.
Using the RNA interference scientists can reduce gene expression using siRNA or shRNA- gene knockdown.
Applications of gene knockout:
One of the important application of gene knockout is to study the function of a particular gene.
It also enables scientists to monitor and control the effect of a gene.
Gene knockout method is used for constructing genetically modified organism such as GM plants, GM bacteria and GM animals.
It is also used to study the effect and contribution of a particular gene and its role in the development of a disease.
It is likewise employed in drug discovery: using gene knockout like genetic engineering tools, drug screening can be done.
One of the major limitations of the gene knockout is missing the target gene. Yet the present method is one of the powerful tool used to study gene function.
In addition to this, gene knockout method is also used in the plant genomic research. Different knockout plant species can be constructed for testing different stress resistance phenotypes.
- Scientists can analyse gene function by deleting the gene sequence. Nature; Essential of genetics unit 4.4 (link).
- Hall B, Limaye A, Kulkarni AB. Overview: generation of gene knockout mice. Curr Protoc Cell Biol. 2009; Chapter 19:Unit–19.12.17.