Elimination of selectable marker genes is essential technique for the relaxation of consumer concerns on genetically modified organisms (GMOs) and gene stacking. In this study, a self-removable selectable marker cassette consists of glucocorticoid-regulated Cre/loxP recombination system, nptii and a negative selection marker cytosine deaminase (codA) gene was constructed between the strong plant promoter CaMV 35S promoter and a β-glucuronidase (GUS) gene. By the induction of cre expression with glucocorticoid hormone dexamethasone, the cassette was eliminated and GUS reporter gene was activated. The eliminated region was confirmed by PCR. Cells still carrying selectable markers were killed by codA that converts non-toxic 5-fluorocytosine to toxic 5-fluorouracil.
식물에서 glucocorticoid 호르몬으로 transcription을 induction할 수 있는 GVG system을 이용해 박테리오파지 P1의 Cre recombinase의 발현을 조절하여 loxP site 사이의 selection marker 유전자들을 형질전환 후 제거할 수 있는 pCAG100 벡터를 만들었다. 담배에 형질전환시켜 kanamycin으로 selection한 후, DEX로 induction하였다. GUS의 활성이 나타나는 것으로 35S promoter와 GUS 사이의 selection marker들이 제거되었음을 확인할 수 있었다. 또한, PCR로 제거 부위를 확인하였다. 그 뒤, selection marker가 제거되지 않은 세포들은 독성이 없는 5-fluorocytosine을 독성이 있는 5-fluorourasil로 바꾸는 cytosine deaminase 유전자를 이용해 사멸시켰다.
