Gene Knockin Technique

What Is Gene Knockin?

A knock in (or gene knock in) refers to a genetic engineering 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 [1]. 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 [2]. The difference between knock in technology and traditional transgenic techniques is that a knock in involves a gene inserted into a specific locus, and is thus a "targeted" insertion.

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

Homology and non-homology based gene insertion mutation
Homology and non-homology based gene insertion mutation

Fig 1. Gene insertion mutation. A. Homology-based gene insertion mutation; B. Non-homology-based gene insertion mutation.

Gene Knockin Technique Contains The Following Sections

Gene Knockin Techniques

Gene knock in originated as a slight modification of the original knockout technique developed by Martin Evans, Oliver Smithies, and Mario Capecchi. 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 [3]. 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 [4].

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 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 carcinogenicity [5].

More recent developments in knock in technique have allowed for pigs to have a gene for green fluorescent protein inserted with a CRISPR/Cas9 system, which allows for much more accurate and successful gene insertions [6]. 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 [7].

Gene Knockin Technique Related References

1. Gibson, Greg (2009). A Primer Of Genome Science 3rd ed. Sunderland, Massachusetts: Sinauer. pp. 301–302.
2. Mouse Genome Sequencing Consortium; Waterston RH et al. (2002-12-05). Initial sequencing and comparative analysis of the mouse genome. Nature. 420 (6915): 520–562.
3. Westphal CH; Leder P (1997). Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice. Curr Biol. 7 (7): 530–533.
4. Manis, John P. (2007-12-13). Knock out, knock in, knock down-genetically manipulated mice and the Nobel Prize. The New England Journal of Medicine. 357 (24): 2426–2429.
5. Liu, Zhipei et al. (2005-04-01). p53 mutations in benzo(a)pyrene-exposed human p53 knock-in murine fibroblasts correlate with p53 mutations in human lung tumors. Cancer Research. 65 (7): 2583–2587.
6. Ruan, Jinxue et al. (2015-09-18). Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific Reports. 5: 14253.
7. Wang Y et al. (2016-02). Highly efficient generation of biallelic reporter gene knock-in mice via CRISPR-mediated genome editing of ESCs. Protein Cell. 7 (2): 152–156.