Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection
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 rep...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-08-01
|
| Series: | Applied Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-3417/12/15/7938 |
| _version_ | 1797414500444405760 |
|---|---|
| author | Marija Đorđević Verica Paunović Maja Jovanović Tucović Anja Tolić Jovana Rajić Svetlana Dinić Aleksandra Uskoković Nevena Grdović Mirjana Mihailović Ivanka Marković Jelena Arambašić Jovanović Melita Vidaković |
| author_facet | Marija Đorđević Verica Paunović Maja Jovanović Tucović Anja Tolić Jovana Rajić Svetlana Dinić Aleksandra Uskoković Nevena Grdović Mirjana Mihailović Ivanka Marković Jelena Arambašić Jovanović Melita Vidaković |
| author_sort | Marija Đorđević |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T05:34:10Z |
| format | Article |
| id | doaj.art-16c02b491d4948f28ad24661373c371b |
| institution | Directory Open Access Journal |
| issn | 2076-3417 |
| language | English |
| last_indexed | 2024-03-09T05:34:10Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Applied Sciences |
| spelling | doaj.art-16c02b491d4948f28ad24661373c371b2023-12-03T12:30:20ZengMDPI AGApplied Sciences2076-34172022-08-011215793810.3390/app12157938Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line TransfectionMarija Đorđević0Verica Paunović1Maja Jovanović Tucović2Anja Tolić3Jovana Rajić4Svetlana Dinić5Aleksandra Uskoković6Nevena Grdović7Mirjana Mihailović8Ivanka Marković9Jelena Arambašić Jovanović10Melita Vidaković11Department of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaInstitute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Dr. Subotića 1, 11000 Belgrade, SerbiaInstitute of Medical and Clinical Biochemistry, Faculty of Medicine, University of Belgrade, Pasterova 2, 11000 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaInstitute of Medical and Clinical Biochemistry, Faculty of Medicine, University of Belgrade, Pasterova 2, 11000 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaDepartment of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, SerbiaAn 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.https://www.mdpi.com/2076-3417/12/15/7938alpha TC1-6 cellstransfectionPEInucleofectiongreen fluorescent proteinEpiCRISPR |
| spellingShingle | Marija Đorđević Verica Paunović Maja Jovanović Tucović Anja Tolić Jovana Rajić Svetlana Dinić Aleksandra Uskoković Nevena Grdović Mirjana Mihailović Ivanka Marković Jelena Arambašić Jovanović Melita Vidaković Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection Applied Sciences alpha TC1-6 cells transfection PEI nucleofection green fluorescent protein EpiCRISPR |
| title | Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection |
| title_full | Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection |
| title_fullStr | Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection |
| title_full_unstemmed | Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection |
| title_short | Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection |
| title_sort | nucleofection as an efficient method for alpha tc1 6 cell line transfection |
| topic | alpha TC1-6 cells transfection PEI nucleofection green fluorescent protein EpiCRISPR |
| url | https://www.mdpi.com/2076-3417/12/15/7938 |
| work_keys_str_mv | AT marijađorđevic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT vericapaunovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT majajovanovictucovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT anjatolic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT jovanarajic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT svetlanadinic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT aleksandrauskokovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT nevenagrdovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT mirjanamihailovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT ivankamarkovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT jelenaarambasicjovanovic nucleofectionasanefficientmethodforalphatc16celllinetransfection AT melitavidakovic nucleofectionasanefficientmethodforalphatc16celllinetransfection |