Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
2022
Аутори:
Đorđević, MarijaPaunović, Verica
Jovanović Tucović, Maja
Tolić, Anja
Rajić, Jovana
Dinić, Svetlana
Uskoković, Aleksandra
Grdović, Nevena
Mihailović, Mirjana
Marković, Ivanka
Arambašić Jovanović, Jelena
Vidaković, Melita
Тип документа:
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт:
An efficient transfection is a crucial step for the introduction of epigenetic modification in host cells, and there is a need for an optimized transfection process for individual model systems separately. Mouse pancreatic αTC1-6 cells, which act as an attractive model system for epigenetic cell reprogramming and diabetes treatment, were transiently transfected with two different transfection methods: the chemical method with polyethyleneimine (PEI) and nucleofection as a physical transfection method. Flow cytometry and fluorescent microscopy examination of GFP expression showed that transfection efficiency was affected by the size of plasmids using both transfection methods. Subsequently, the Cas9 mRNA expression confirmed successful transfection with EpiCRISPR plasmid, whereas the cell physiology remained unchanged. The adjusted nucleofection protocol for αTC1-6 cells transfected with an EpiCRISPR mix of plasmids reached 71.1% of GFP-positive transfected cells on the fifth post-transfection day and proved to be much more efficient than the 3.8% GFP-positive PEI transfected cells. Modifying the protocol, we finally specify CM-156 program and SF 4D-Nucleofector X Solutions for Amaxa™ nucleofection as a method of choice for alpha TC1-6 cell line transfection.
Кључне речи:
alpha TC1-6 cells; EpiCRISPR; green fluorescent protein; nucleofection; PEI; transfectionИзвор:
Applied Sciences, 2022, 12, 15, 7938-Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200007 (Универзитет у Београду, Институт за биолошка истраживања 'Синиша Станковић') (RS-MESTD-inst-2020-200007)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200110 (Универзитет у Београду, Медицински факултет) (RS-MESTD-inst-2020-200110)
- European Cooperation in Science and Technology (COST)
DOI: 10.3390/app12157938
ISSN: 2076-3417