Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy.

Autor: Mlakar V; CANSEARCH Research Platform for Pediatric Oncology and Hematology, Department of Pediatrics, Gynecology and Obstetrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland. vid.mlakar@unige.ch., Lesne L; CANSEARCH Research Platform for Pediatric Oncology and Hematology, Department of Pediatrics, Gynecology and Obstetrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland., Vossio S; University of Geneva, School of Chemistry and Biochemistry - Sciences II, ACCESS Geneva, Quai Ernest Ansermet 30, 1211, Geneva 4, Switzerland., Dupanloup I; CANSEARCH Research Platform for Pediatric Oncology and Hematology, Department of Pediatrics, Gynecology and Obstetrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.; Swiss Institute of Bioinformatics, Lausanne, Switzerland., Gloor Y; CANSEARCH Research Platform for Pediatric Oncology and Hematology, Department of Pediatrics, Gynecology and Obstetrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland., Moreau D; Swiss Institute of Bioinformatics, Lausanne, Switzerland., Ansari M; CANSEARCH Research Platform for Pediatric Oncology and Hematology, Department of Pediatrics, Gynecology and Obstetrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.; Division of Pediatric Oncology and Hematology, Department of Women, Child and Adolescent, University Geneva Hospitals, Geneva, Switzerland.
Jazyk: angličtina
Zdroj: BMC biotechnology [BMC Biotechnol] 2024 Oct 15; Vol. 24 (1), pp. 81. Date of Electronic Publication: 2024 Oct 15.
DOI: 10.1186/s12896-024-00911-z
Abstrakt: Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don't tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.
(© 2024. The Author(s).)
Databáze: MEDLINE
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