Dr. S.T. Jiang’s CV

Curriculum Vitae jiang4(1)

When CRISPR/cas9 meet BAC in mouse:
integrated genome-modification technologies

CRISPR/cas9 基因編輯技術近年已廣泛應用於基因改造小鼠的產製,尤其是基因標的小鼠,以往要利用小鼠胚幹細胞及嵌合鼠產製的昂貴耗時流程,目前幾乎已被淘汰,皆改以此新技術,大大降低生產成本與時程,使許多科學家紛紛開始利用基改鼠做研究;但此基因編輯技術目前只能做基因小片段移除藉以破壞基因轉譯碼,或是藉由一段外來單股DNA來置換特定鹼基以造成基因的點突變,局限性仍大。為了擴展其應用,我們利用人工細菌染體(Bacterial Artificial Chromosome: BAC)的高承載DNA特性,將實驗設計一一載入其中,包括時間、空間、劑量控制介面,或是多個基因同時參與等,皆可依需求載入,最後再以CRISPR/cas9技術定點插入所要標的之基因位置,以完成整合式基改的目標,以單一基改動物完成多功能性的實驗需求,達到減量使用實驗動物及省時省錢的目標。

The CRISPR/cas9 gene editing technique is widely used for gene-modification (GM) mouse production especially for the gene-targeting in recent years. In the past, the gene targeting was employed in mouse embryonic stem cells first and then transfer to blastocysts so as to generate chimera. The production cost is about 30,000 USD, and usually takes more than one year to get the targeting mouse. Now using the gene editing technique dramatically costs down to about 5,000 USD, and takes only 6 months to touch down. So lots of scientists are willing to create GM mice as research models recently. But the limitation of the new technique is the DNA length for gene editing, most of insertion or deletion (INDELs) are 2-16 bp in length. For the generation of mice with a point mutation at specific genes, a single strand template DNA with desired point mutation could be applied in the editing technique to achieve this goal. It’s still difficult to knock in long DNA fragments via a homology-directed repair. To break through the bottleneck, we introduced the bacterial artificial chromosome (BAC) as the homologous targeting template in the gene editing system to integrate multiple gene controlling elements including spatial, temporal, and dosage control in one construct. By the way, we could generate a GM mouse with multiple functions fitting in specific experimental designing in order to achieve 3Rs principles and save time and money synonym.

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