Principle and method of gene knockout
Gene knockout can be said to be a combination of genomics, cell isolation and culture, and transgenic technology. So what is the principle of gene knockout? What are the methods of gene knockout? Here, make a summary for everyone to learn.
I. Overview:
Gene knockout is a new type of molecular biology technology developed since the late 1980s. It is a technology that inactivates or deletes specific genes in the body through certain methods. Gene knockout in the general sense mainly uses the principle of DNA homologous recombination, and replaces target gene fragments with designed homologous fragments, so as to achieve the purpose of gene knockout. With the development of gene knockout technology, in addition to homologous recombination, new principles and techniques have also been gradually applied. More successful gene insertion mutations and iRNAs can also achieve the purpose of gene knockout.
2. Various principles and methods for gene knockout:
1. Gene homologous recombination for gene knockout
Gene knockout was developed in the second half of the 1980s using the principle of DNA homologous recombination. In the early 1980s, the successful isolation and in vitro culture of embryonic stem cells (ES cells) laid the technical foundation for gene knockout. In 1985, the first confirmed existence of homologous recombination in mammalian cells laid the theoretical foundation for gene knockout. By 1987, Thompsson first established a complete mouse model of ES cell gene knockout [1]. Until now, the use of gene homologous recombination for gene knockout is still the most common method used in the construction of gene knockout animal models.
(1) Basic steps for constructing a gene knockout animal model using homologous recombination:
â‘ . Construction of gene vector: The target gene and the DNA molecule homologous to the specific fragment of the target gene in the cell are recombined into a vector with marker genes (such as neo gene, TK gene, etc.) to become a recombinant vector. Gene knockout is to make a gene lose its physiological function, so it is generally designed as a replacement vector.
â‘¡. ES cell acquisition: At present, gene knockout is generally used as embryonic stem cells, the most commonly used is mouse, and embryonic stem cells such as rabbits, pigs and chickens are also used. The commonly used mouse germline is 129 and its hybrids, because these mice have a tendency to spontaneous mutations to form teratomas and teratosarcomas, which is an ideal experimental animal for gene knockout. Embryonic stem cell lines of other genetic backgrounds are gradually being developed and applied.
â‘¢. Homologous recombination: Recombination vectors are introduced into homologous embryonic stem cells (ES cells) by a certain method (electroporation or microinjection), so that homologous recombination of foreign DNA and corresponding parts of the embryonic stem cell genome occurs, Integrate the DNA sequence in the recombinant vector into the endogenous genome to be expressed. Generally, microinjection has a higher hit rate, but the technical difficulty is greater. Electroporation hit rate is lower than microinjection, but it is easy to use.
â‘£. Select the cells that have been hit: due to the extremely low natural rate of homologous recombination of gene transfer, the recombination probability of animals is 10-2 ~ 10-5, and the probability of plants is 10-4 ~ 10-5. Therefore, it is very important to screen embryonic stem cells that have undergone homologous recombination from many cells. At present, the commonly used methods are positive and negative screening method (PNS method), specific gene expression method of marker gene and PCR method. The PNS method is the most widely used.
⑤. Phenotype research: By observing the changes in the biological shape of chimeras and then understanding the changes in the biological shape of the mice before and after the change of the target gene, the purpose of studying the target gene can be achieved.
â‘¥. Obtaining homozygote: Since homologous recombination often occurs on one chromosome of a pair of chromosomes, if you want to obtain a stable inherited homozygous gene knockout model, at least two generations of inheritance are required.
(2) Conditional gene knockout
Conditional gene knockout can be defined as a special gene knockout method that restricts the modification of a gene to certain types of cells or a specific stage of development in mice [2]. It is actually based on the conventional gene knockout, using the site-specific recombination technology mediated by the recombinase Cre to set a controllable "button" on the temporal and spatial range of gene modification in mice, so that The range and time of modification of the mouse genome is in a controlled state.
The principle of conditional knockout:
Using Cre / LoxP and the FLP-frt system from yeast can study the phenotype caused by the inactivation of target genes in specific tissues or specific cells [7]. Use conventional gene targeting to install two 1oxPs arranged in the same direction on the target site of the genome, and attach loxP ("loxP floxed") ES cells on both sides to generate "loxPfloxed" mice. Then, by " loxP floxed "mice are crossed with Cre transgenic mice (Cre recombinase can also be introduced into the mice in other ways) to produce conditionally mutated mice in which the target genes are modified in specific ways (eg, specific tissue specificity). In "loxP floxed" mice, although a loxP has been installed on both sides of the target gene, the target gene has no other changes, so the "1oxP noxed" mouse phenotype is still the same as the wild type. But when it is crossed with Cre transgenic mice, the resulting offspring will carry both the "loxP floxed" target gene and the Cre gene. Cre recombinase produced by Cre gene expression will mediate the excision reaction between 1oxP on both sides of the target gene, resulting in the excision of a loxP and the target gene. In this way, the modification (excision) of the target gene is premised on the expression of Cre. The expression characteristics of Cre determine the persistence of target gene modification (excision): that is, in which tissue cell Cre is expressed, the target gene modification (excision) occurs in which tissue cell; and the expression level of Cre will affect the target The efficiency of gene modification in such tissue cells. So as long as the specificity and level of Cre expression are controlled, the specificity and degree of target gene modification in mice can be achieved
(3) Inducible gene knockout
Inducible gene knockout is also based on the Cre / loxp system, but it uses the activity of the promoter that controls Cre expression or the activity of the expressed Cre enzyme to have inducible characteristics. By controlling the time of the inducing agent or using Cre The host cell specificity of the vector in the gene localization expression system and the temporal controllability of the process of transferring the expression system into the animal, thereby achieving genetic modification of specific genes in certain developmental stages and certain tissue cells of 1oxP The purpose of the gene knockout technology. One can artificially control the spatiotemporal specificity of animal gene mutations in a pre-designed way to give the inducer time to avoid the phenomenon of stillbirth or death of the animal shortly after birth. Several common inducible types are as follows: tetracycline-inducible (Figure 4); interferon-inducible; hormone-inducible; adenovirus-mediated.
Advantages of inducible gene knockout:
â‘ The time to induce gene mutation can be controlled artificially;
â‘¡ Can avoid the problem of stillbirth due to gene mutation
â‘¢ The recombination rate between 2 loxP sites is high;
â‘£ If a gene transfer system such as a virus or ligand / DNA complex is used to mediate the expression of Cre, the process of establishing Cre-transgenic animals can be omitted. [10]
2.2 Gene knockout using random insertion mutations.
2.2.1 Principle:
This method uses certain viruses, bacteria, or other gene vectors that can randomly insert gene sequences, perform random insertion mutations in the target cell genome, establish a cell bank carrying random insertion mutations, and then screen through the corresponding markers to obtain the corresponding genes Knock out cells (see Figure 5 for the principle) [11,12]. Depending on the cell, the choice of inserting the vector is also different. Reverse rate viruses can be used for the insertion of animal and plant cells; for plant cells, Agrobacterium-mediated T-DNA transformation and transposons are more commonly used; bacteriophages can be used for bacterial gene knockout.
2.2.2 Gene capture method
The gene capture method is a new method developed recently that uses random insertion mutations for gene knockout. The principle can be seen in Figure 6. Gene capture vectors usually also include a promoter-less reporter gene, usually a neo gene. The neo gene is inserted into the ES cell genome and the transcriptional regulatory elements of the captured gene are used to achieve expression. ES clones can be easily contained in G418-containing Screened in the selection medium, in theory, clones that survive in the selection medium should contain 100% of the target gene. The target gene information can be obtained by screening marker gene flanking cDNA or genomic sequence analysis [13]
2.2.3 Advantages and disadvantages of gene capture method
Gene knockout research using conventional methods takes a lot of time and manpower. Researchers must screen relevant genomic clones in the genomic library for the target site, draw corresponding physical maps, construct specific gene knockout vectors Screening of target ES cells, etc., usually takes one year or more to obtain a knockout homozygous mouse. In the face of the huge unknown genetic information generated by the Human Genome Project, the traditional gene knockout method seems a bit powerless. Therefore, the gene capture method came into being. Using gene capture can establish an ES cell library carrying random insertion mutations, saving a lot of work and costs for screening genomic libraries and constructing specific targeting vectors, and more efficiently and quickly carrying out mouse chromosomes. Functional analysis of the group.
The disadvantage of this method is that it can only delete genes expressed in Es cells. The number of genes expressed in a single cell type is about I04. The current gene capture vector should theoretically be able to delete all genes expressed in ES cells. Therefore, gene capture in ES cells is still promising. Another disadvantage of gene knockout by gene capture is that it is impossible to finely modify genes,
2.3. Gene knockout caused by RNAi.
Since a small amount of double-stranded RNA can block gene expression, and this effect can be transmitted to the progeny cells, the RNAi reaction process can also be used for gene knockout. In recent years, more and more gene knockouts have adopted RNAi, a simpler and more convenient method.
2.3.1 The mechanism of RNAi blocking gene expression
After double-stranded RNA enters the cell, it can be cleaved into siRNA under the action of Dicer enzyme. On the other hand, double-stranded RNA can also be used in RdRP (RNA-directed RNA polymerase using RNA as template to guide RNA synthesis
After polymerase (RdRP) self-amplifies, it is cleaved into siRNA by Dicer enzyme. The double-stranded SiRNA becomes single-stranded and forms a complex with certain proteins. Argonaute2 is currently the only known protein involved in complex formation. This complex binds to mRNA complementary to siRNA. On the one hand, the mRNA is cleaved by RNase. On the other hand, SiRNA is used as a primer and mRNA is used as a template to synthesize the complementary strand of mRNA under the action of RdRP. As a result, the mRNA also becomes double-stranded RNA, which is also cleaved into siRNA under the action of Dicer enzyme. These newly generated siRNAs also have the effect of inducing RNAi. Through this polymerase chain reaction, the intracellular siRNA is greatly increased, significantly increasing the suppression of gene expression. RNAi from 21 to 23 nucleotides to double-stranded RNA of several hundred nucleotides can induce RNAi, but the effect of long double-stranded RNA in blocking gene expression is significantly stronger than that of short double-stranded RNA [13].
2.3.2 Advantages and applications of RNAi gene knockout
â‘ . It is more convenient than the homologous recombination method, and the cycle is greatly shortened.
â‘¡. For mammals, such as some genes that will die when the mouse is embryonic after knockout, RNAi technology can be used to study its function in cells cultured in vitro.
â‘¢. Because RNAi can efficiently and specifically block gene expression, it has become a good tool for studying signal transduction pathways.
â‘£. RNAi is also used to study genes that play a role in development. For example, RNAi can be used to block the expression of certain genes to study whether they play a key role in the proliferation and differentiation of embryonic stem cells.
2.4 Other principles for gene knockout.
In addition to the above-mentioned mature and generally used gene knockout principles, there are some knockout technologies based on other principles that are in the process of research and improvement, such as gene knockout guided by TFOs (Triple helix forming oligonucleotides) [16] and the application of antisense technology in gene knockout technology, etc. [17]. With the development of genetics and molecular biology theory, new gene knockout principles are constantly being discovered and explored.
3. Application and prospects of gene knockout technology:
â‘ . Establish a biological model. The establishment of model organisms is very important in the study of gene function and metabolic pathways. Gene knockout technology is often used to establish a biological model of a specific gene deletion, so as to conduct related research. These models can be cells or complete animals, plants, or microorganisms. The most common are mice, and gene knockout models of rabbits, pigs, nematodes, yeast, and Arabidopsis are also commonly reported.
â‘¡. Research on the molecular mechanism of diseases and gene therapy of diseases. Gene knockout technology can determine the nature of a specific gene and study its impact on the body. This is of great significance for understanding the root of the disease or finding the target of gene therapy.
③. Provide cheap xenotransplanted organs. As we all know, the scarcity of organ sources is often a major constraint for human organ transplantation, but a large number of cheap xenobiotic organs such as pigs cannot be used in the human body. This is because the genes of heterologous organisms will produce some heterologous molecules that can cause strong immune rejection in the human body. If the genes that produce these heterologous molecules can be knocked out, the animal ’s organs can be used for the treatment of human diseases, which will Bring a great gospel to patients. For example, PPL Therapeutics has successfully knocked out the α-1,3GT gene using gene knockout technology in pig somatic cells in 1999. Each pig is deficient in 2 copies of the gene producing a1-3 galactosyltransferase. These enzymes produce a sugar molecule on the cell surface, and the body's immune system can immediately recognize this sugar molecule as heterogeneous, thereby triggering a hyperacute immune rejection reaction. In the absence of this enzyme, hyperacute rejection will not occur again [10].
â‘£. Application in immunology. Similar to heterologous organ transplantation, when heterologous antibodies are used in humans, there will be more or less immune rejection, which hinders the production and application of human antibody drugs. If the animal immune molecule gene is knocked out and replaced with the corresponding human gene, then human antibodies will be produced, thereby solving the problem of human antibody production.
⑤ Transform organisms and cultivate new species. Bacterial genetic engineering technology is a major breakthrough in the history of molecular biology this century, and gene knockout technology may be another major leap in genetic engineering. It provides important technical support for the targeted transformation of organisms and the cultivation of new organisms.
4. Defects of gene knockout technology
With the development of gene knockout technology, many shortcomings and defects in early technology have been solved, but gene knockout technology always has an insurmountable shortcoming, that is, knocking out a gene does not necessarily know the function of the gene , The reasons include: on the one hand, many genes are functionally redundant, knock out one
Functionally redundant genes do not result in an easily recognizable phenotype, because other members of the gene family can provide the same function; on the other hand, for certain essential genes, knocking out will cause cell lethality, also You ca n’t study these essential genes.
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