Knock-In Mice Protocol

New Methods of Using Previously Failed Knock-in Mouse Protocols to Gain Higher Efficiency

The importance of knock-in mice in genetic research cannot be undervalued. With so many genetic researchers relying on data from knock-in mouse models to determine whether or not a certain treatment, technique, or drug may be effective for countering various disorders, new methods of achieving knock-in mouse models have also been developed. CRISPR/Cas9, a method that was previously believed to only work for generating knock-out mouse models, has been re-evaluated recently with some favorable results. Even though the success rate of most CRISPR knock-in protocols was below 10%, researchers are now realizing that the percentage could be higher by using a CRISPR protocol that was discovered through the testing of long, single-stranded DNAs serving as donors.

A knock-in gene refers to a method of genetic engineering that involves the one-for-one substitution of a DNA sequence found at a specific locus, with another sequence. In some cases, the replacement sequence isn’t actually present within the locus, as it’s foreign to the organism. Gene knock-ins have been used and refined for a long time. Now, with the advent of genetic technology, it is possible to use increasingly more complicated, intricate and efficient knock-in methods, in order to achieve the desired result. Knock in mouse models have been used primarily for the purpose of creating disease models, allowing researchers to test how a certain gene or genetically induced disease behaves from birth to death, what medicines and drugs can be used for treatment, and what genetic mechanism fuels its progress.

The most common technique for using gene knock-ins involves a slight change in the original knock-out protocol invented by Oliver Smithies, Martin Evans, and Mario Capecchi. Sometimes the modifications are introduced with the help of stem cells, while other times the mutated copy of a p53 gene will allow them to be inserted into the mouth genome. The recent improvements in knock-in gene creation due to CRISPR, however, have attracted the most recent spotlight. Although the efficiency is still not comparable to stem cells, the world of science is slowly recognizing the success of this CRISPR and aims to use it more in the near future. Regardless of the method used, a gene knock-in is regarded as a gain of function mutation, which means the body gains an added function aside from the ones that its DNA was already capable of. As a result, gene knock-ins in mice can be used to more accurately study genetic mutations in humans.

Using CRISPR/Cas9 to obtain genetic mutations through knock-in mice is not a new concept. However, previously there were very few positive results, and most knock-in mice obtained with this technique died. With the use of long, single stranded DNA sources, however, it has been established that the CRISPR/Cas9 technique can be brought from a success rate of 1% to 10%, to higher than 10%. For obtaining knock-in mice, single-stranded donors have proven to be extremely efficient, both in the case of insertion and gene replacement. However, further research will be needed for considering the future success in generating knock-in mice. Most experts now believe it is a viable solution.

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