molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...

and

biology Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary i ...

, a gene knock-in (abbreviation: KI) refers to a

genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...

method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus. Typically, this is done in mice since the technology for this process is more refined and there is a high degree of shared sequence complexity between mice and humans. The difference between knock-in technology and traditional

transgenic A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...

techniques is that a knock-in involves a gene inserted into a specific locus, and is thus a "targeted" insertion. It is the opposite of

gene knockout A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene kno ...

. A common use of knock-in technology is for the creation of disease models. It is a technique by which scientific investigators may study the function of the regulatory machinery (e.g. promoters) that governs the expression of the natural gene being replaced. This is accomplished by observing the new phenotype of the organism in question. The BACs and YACs are used in this case so that large fragments can be transferred.

Technique

Gene knock-in originated as a slight modification of the original knockout technique developed by

Martin Evans Sir Martin John Evans (born 1 January 1941) is an English biologist who, with Matthew Kaufman, was the first to culture mice embryonic stem cells and cultivate them in a laboratory in 1981. He is also known, along with Mario Capecchi and Olive ...

Oliver Smithies Oliver Smithies (23 June 1925 – 10 January 2017) was a British-American geneticist and physical biochemist. He is known for introducing starch as a medium for gel electrophoresis in 1955, and for the discovery, simultaneously with Mario Capec ...

, and

Mario Capecchi Mario Ramberg Capecchi (born 6 October 1937) is an Italian-born molecular geneticist and a co-awardee of the 2007 Nobel Prize in Physiology or Medicine for discovering a method to create mice in which a specific gene is turned off, known as knoc ...

. Traditionally, knock-in techniques have relied on homologous recombination to drive targeted gene replacement, although other methods using a transposon-mediated system to insert the target gene have been developed. The use of ''loxP'' flanking sites that become excised upon expression of Cre recombinase with gene vectors is an example of this. Embryonic stem cells with the modification of interest are then implanted into a viable blastocyst, which will grow into a mature chimeric mouse with some cells having the original blastocyst cell genetic information and other cells having the modifications introduced to the embryonic stem cells. Subsequent offspring of the chimeric mouse will then have the gene knock-in. Gene knock-in has allowed, for the first time, hypothesis-driven studies on gene modifications and resultant phenotypes. Mutations in the human p53 gene, for example, can be induced by exposure to benzo(a)pyrene (BaP) and the mutated copy of the p53 gene can be inserted into mouse genomes. Lung tumors observed in the knock-in mice offer support for the hypothesis of BaP’s

carcinogen A carcinogen is any substance, radionuclide, or radiation that promotes carcinogenesis (the formation of cancer). This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes. Several radioactive substan ...

icity. More recent developments in knock-in technique have allowed for pigs to have a gene for green fluorescent protein inserted with a

CRISPR/Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic e ...

system, which allows for much more accurate and successful gene insertions. The speed of CRISPR/Cas9-mediated gene knock-in also allows for biallelic modifications to some genes to be generated and the phenotype in mice observed in a single generation, an unprecedented timeframe.

Versus gene knockout

Knock-in technology is different from

knockout A knockout (abbreviated to KO or K.O.) is a fight-ending, winning criterion in several full-contact combat sports, such as boxing, kickboxing, muay thai, mixed martial arts, karate, some forms of taekwondo and other sports involving striking, a ...

technology in that knockout technology aims to either delete part of the DNA sequence or insert irrelevant DNA sequence information to disrupt the expression of a specific genetic locus. Gene knock-in technology, on the other hand, alters the genetic locus of interest via a one-for-one substitution of DNA sequence information or by the addition of sequence information that is not found on said genetic locus. A gene knock-in therefore can be seen as a gain of function mutation and a gene knockout a loss of function mutation, but a gene knock-in may also involve the substitution of a functional gene locus for a mutant phenotype that results in some loss of function.

Potential applications

Because of the success of gene knock-in methods thus far, many clinical applications can be envisioned. Knock-in of sections of the human

immunoglobulin gene An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

into mice has already been shown to allow them to produce humanized antibodies that are therapeutically useful. It should be possible to modify

stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...

s in humans to restore targeted gene function in certain tissues, for example possibly correcting the mutant gamma-chain gene of the

IL-2 receptor The interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds and responds to a cytokine called IL-2. Composition IL-2 binds to the IL-2 receptor, which ha ...

in hematopoietic stem cells to restore lymphocyte development in people with X-linked

severe combined immunodeficiency Severe combined immunodeficiency (SCID), also known as Swiss-type agammaglobulinemia, is a rare genetic disorder characterized by the disturbed development of functional T cells and B cells caused by numerous genetic mutations that result in diffe ...

Limitations

While gene knock-in technology has proven to be a powerful technique for the generation of models of human disease and insight into proteins ''in vivo'', numerous limitations still exist. Many of these are shared with the limitations of knockout technology. First, combinations of knock-in genes lead to growing complexity in the interactions that inserted genes and their products have with other sections of the genome and can therefore lead to more side effects and difficult-to-explain

phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...

s. Also, only a few loci, such as the ROSA26 locus have been characterized well enough where they can be used for conditional gene knock-ins; making combinations of

reporter A journalist is an individual that collects/gathers information in form of text, audio, or pictures, processes them into a news-worthy form, and disseminates it to the public. The act or process mainly done by the journalist is called journalism ...

and transgenes in the same locus problematic. The biggest disadvantage of using gene knock-in for human disease model generation is that mouse physiology is not identical to that of humans and human

ortholog Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spec ...

s of proteins expressed in mice will often not wholly reflect the role of a gene in human pathology. This can be seen in mice produced with the

ΔF508 Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein and anion channel in vertebrates that is encoded by the ''CFTR'' gene. Geneticist Lap-Chee Tsui and his team identified the CFTR gene in 1989 as the gene linked wit ...

fibrosis mutation in the CFTR gene, which accounts for more than 70% of the mutations in this gene for the human population and leads to cystic fibrosis. While ΔF508 CF mice do exhibit the processing defects characteristic of the human mutation, they do not display the pulmonary pathophysiological changes seen in humans and carry virtually no lung phenotype. Such problems could be ameliorated by the use of a variety of animal models, and pig models (pig lungs share many biochemical and physiological similarities with human lungs) have been generated in an attempt to better explain the activity of the ΔF508 mutation.

References

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External links