An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase
Background and purpose: Aspartyl/asparaginyl β-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfecti...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wolters Kluwer Medknow Publications
2020-01-01
|
| Series: | Research in Pharmaceutical Sciences |
| Subjects: | |
| Online Access: | http://www.rpsjournal.net/article.asp?issn=1735-5362;year=2020;volume=15;issue=3;spage=291;epage=299;aulast=Bakhtiari |
| _version_ | 1819082473552740352 |
|---|---|
| author | Hadi Bakhtiari Abbas Ali Palizban Hossein Khanahmad Mohammad Reza Mofid |
| author_facet | Hadi Bakhtiari Abbas Ali Palizban Hossein Khanahmad Mohammad Reza Mofid |
| author_sort | Hadi Bakhtiari |
| collection | DOAJ |
| description | Background and purpose: Aspartyl/asparaginyl β-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfection, the obtained cells normally contain a range of cells expressing the different levels of the target of interest. In this paper, we report on our simple innovative approach in the selection of best-transfected cells with the highest expression of ASPH using subclone selection, quantitative real-time polymerase chain reaction, and gradual increment of hygromycin concentration.
Experimental approach: To achieve this goal, human embryonic kidney (HEK 293T) cells were transfected with an ASPH-bearing pcDNA3.1/Hygro(+) vector. During antibiotic selection, single accumulations of the resistant cells were separately cultured and the ASPH mRNA levels of each flask were evaluated. The best subclones were treated with a gradually increasing amount of hygromycin. The ASPH protein expression of the obtained cells was finally evaluated using flow cytometry and immunocytochemistry.
Findings / Results: The results showed that different selected subclones expressed different levels of ASPH. Furthermore, the gradual increment of hygromycin (up to 400mg/mL) improved the expression of ASPH. The best relative fold change in mRNA levels was 57.59 ± 4.11. Approximately 90.2% of HEKASPH cells overexpressed ASPH on their surface.
Conclusion and implications: The experiments indicated that we have successfully constructed and evaluated a recombinant human cell line overexpressing ASPH on the surface. Moreover, our innovative selection approach provided an effective procedure for enriching highly expressing recombinant cells. |
| first_indexed | 2024-12-21T20:17:14Z |
| format | Article |
| id | doaj.art-365f24014b8c4eb5b9279ad4ac494a61 |
| institution | Directory Open Access Journal |
| issn | 1735-5362 1735-9414 |
| language | English |
| last_indexed | 2024-12-21T20:17:14Z |
| publishDate | 2020-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | Research in Pharmaceutical Sciences |
| spelling | doaj.art-365f24014b8c4eb5b9279ad4ac494a612022-12-21T18:51:35ZengWolters Kluwer Medknow PublicationsResearch in Pharmaceutical Sciences1735-53621735-94142020-01-0115329129910.4103/1735-5362.288436An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylaseHadi BakhtiariAbbas Ali PalizbanHossein KhanahmadMohammad Reza MofidBackground and purpose: Aspartyl/asparaginyl β-hydroxylase (ASPH) is abundantly expressed in malignant neoplastic cells. The establishment of a human cell line overexpressing ASPH could provide the native-like recombinant protein needed for developing theranostic probes. In the process of transfection, the obtained cells normally contain a range of cells expressing the different levels of the target of interest. In this paper, we report on our simple innovative approach in the selection of best-transfected cells with the highest expression of ASPH using subclone selection, quantitative real-time polymerase chain reaction, and gradual increment of hygromycin concentration. Experimental approach: To achieve this goal, human embryonic kidney (HEK 293T) cells were transfected with an ASPH-bearing pcDNA3.1/Hygro(+) vector. During antibiotic selection, single accumulations of the resistant cells were separately cultured and the ASPH mRNA levels of each flask were evaluated. The best subclones were treated with a gradually increasing amount of hygromycin. The ASPH protein expression of the obtained cells was finally evaluated using flow cytometry and immunocytochemistry. Findings / Results: The results showed that different selected subclones expressed different levels of ASPH. Furthermore, the gradual increment of hygromycin (up to 400mg/mL) improved the expression of ASPH. The best relative fold change in mRNA levels was 57.59 ± 4.11. Approximately 90.2% of HEKASPH cells overexpressed ASPH on their surface. Conclusion and implications: The experiments indicated that we have successfully constructed and evaluated a recombinant human cell line overexpressing ASPH on the surface. Moreover, our innovative selection approach provided an effective procedure for enriching highly expressing recombinant cells.http://www.rpsjournal.net/article.asp?issn=1735-5362;year=2020;volume=15;issue=3;spage=291;epage=299;aulast=Bakhtiaricancer; aspartyl/asparaginyl β-hydroxylase; human embryonic kidney (hek293t) cell line; transfection; overexpression |
| spellingShingle | Hadi Bakhtiari Abbas Ali Palizban Hossein Khanahmad Mohammad Reza Mofid An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase Research in Pharmaceutical Sciences cancer; aspartyl/asparaginyl β-hydroxylase; human embryonic kidney (hek293t) cell line; transfection; overexpression |
| title | An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase |
| title_full | An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase |
| title_fullStr | An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase |
| title_full_unstemmed | An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase |
| title_short | An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase |
| title_sort | innovative cell selection approach in developing human cells overexpressing aspartyl asparaginyl β hydroxylase |
| topic | cancer; aspartyl/asparaginyl β-hydroxylase; human embryonic kidney (hek293t) cell line; transfection; overexpression |
| url | http://www.rpsjournal.net/article.asp?issn=1735-5362;year=2020;volume=15;issue=3;spage=291;epage=299;aulast=Bakhtiari |
| work_keys_str_mv | AT hadibakhtiari aninnovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT abbasalipalizban aninnovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT hosseinkhanahmad aninnovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT mohammadrezamofid aninnovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT hadibakhtiari innovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT abbasalipalizban innovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT hosseinkhanahmad innovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase AT mohammadrezamofid innovativecellselectionapproachindevelopinghumancellsoverexpressingaspartylasparaginylbhydroxylase |