Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines
Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straig...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Tomás Suchý [verfasserIn] Isabell Kaczmarek [verfasserIn] Tomislav Maricic [verfasserIn] Christian Zieschang [verfasserIn] Torsten Schöneberg [verfasserIn] Doreen Thor [verfasserIn] Ines Liebscher [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2021 |
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| Schlagwörter: |
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| Übergeordnetes Werk: |
In: Adipocyte - Taylor & Francis Group, 2019, 10(2021), 1, Seite 631-645 |
|---|---|
| Übergeordnetes Werk: |
volume:10 ; year:2021 ; number:1 ; pages:631-645 |
| Links: |
Link aufrufen |
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| DOI / URN: |
10.1080/21623945.2021.1990480 |
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| Katalog-ID: |
DOAJ074166905 |
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| 520 | |a Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. | ||
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10.1080/21623945.2021.1990480 doi (DE-627)DOAJ074166905 (DE-599)DOAJ55f47f5971ea489b9fdb752081c1730e DE-627 ger DE-627 rakwb eng RC648-665 QH573-671 QP1-981 Tomás Suchý verfasserin aut Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. adipocytes cas9 crispr/cas adipocyte function 3t3-l1 genetic knock-out Diseases of the endocrine glands. Clinical endocrinology Cytology Physiology Isabell Kaczmarek verfasserin aut Tomislav Maricic verfasserin aut Christian Zieschang verfasserin aut Torsten Schöneberg verfasserin aut Doreen Thor verfasserin aut Ines Liebscher verfasserin aut In Adipocyte Taylor & Francis Group, 2019 10(2021), 1, Seite 631-645 (DE-627)726123374 (DE-600)2681769-X 2162397X nnns volume:10 year:2021 number:1 pages:631-645 https://doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/article/55f47f5971ea489b9fdb752081c1730e kostenfrei http://dx.doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/toc/2162-3945 Journal toc kostenfrei https://doaj.org/toc/2162-397X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2021 1 631-645 |
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10.1080/21623945.2021.1990480 doi (DE-627)DOAJ074166905 (DE-599)DOAJ55f47f5971ea489b9fdb752081c1730e DE-627 ger DE-627 rakwb eng RC648-665 QH573-671 QP1-981 Tomás Suchý verfasserin aut Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. adipocytes cas9 crispr/cas adipocyte function 3t3-l1 genetic knock-out Diseases of the endocrine glands. Clinical endocrinology Cytology Physiology Isabell Kaczmarek verfasserin aut Tomislav Maricic verfasserin aut Christian Zieschang verfasserin aut Torsten Schöneberg verfasserin aut Doreen Thor verfasserin aut Ines Liebscher verfasserin aut In Adipocyte Taylor & Francis Group, 2019 10(2021), 1, Seite 631-645 (DE-627)726123374 (DE-600)2681769-X 2162397X nnns volume:10 year:2021 number:1 pages:631-645 https://doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/article/55f47f5971ea489b9fdb752081c1730e kostenfrei http://dx.doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/toc/2162-3945 Journal toc kostenfrei https://doaj.org/toc/2162-397X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2021 1 631-645 |
| allfields_unstemmed |
10.1080/21623945.2021.1990480 doi (DE-627)DOAJ074166905 (DE-599)DOAJ55f47f5971ea489b9fdb752081c1730e DE-627 ger DE-627 rakwb eng RC648-665 QH573-671 QP1-981 Tomás Suchý verfasserin aut Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. adipocytes cas9 crispr/cas adipocyte function 3t3-l1 genetic knock-out Diseases of the endocrine glands. Clinical endocrinology Cytology Physiology Isabell Kaczmarek verfasserin aut Tomislav Maricic verfasserin aut Christian Zieschang verfasserin aut Torsten Schöneberg verfasserin aut Doreen Thor verfasserin aut Ines Liebscher verfasserin aut In Adipocyte Taylor & Francis Group, 2019 10(2021), 1, Seite 631-645 (DE-627)726123374 (DE-600)2681769-X 2162397X nnns volume:10 year:2021 number:1 pages:631-645 https://doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/article/55f47f5971ea489b9fdb752081c1730e kostenfrei http://dx.doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/toc/2162-3945 Journal toc kostenfrei https://doaj.org/toc/2162-397X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2021 1 631-645 |
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10.1080/21623945.2021.1990480 doi (DE-627)DOAJ074166905 (DE-599)DOAJ55f47f5971ea489b9fdb752081c1730e DE-627 ger DE-627 rakwb eng RC648-665 QH573-671 QP1-981 Tomás Suchý verfasserin aut Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. adipocytes cas9 crispr/cas adipocyte function 3t3-l1 genetic knock-out Diseases of the endocrine glands. Clinical endocrinology Cytology Physiology Isabell Kaczmarek verfasserin aut Tomislav Maricic verfasserin aut Christian Zieschang verfasserin aut Torsten Schöneberg verfasserin aut Doreen Thor verfasserin aut Ines Liebscher verfasserin aut In Adipocyte Taylor & Francis Group, 2019 10(2021), 1, Seite 631-645 (DE-627)726123374 (DE-600)2681769-X 2162397X nnns volume:10 year:2021 number:1 pages:631-645 https://doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/article/55f47f5971ea489b9fdb752081c1730e kostenfrei http://dx.doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/toc/2162-3945 Journal toc kostenfrei https://doaj.org/toc/2162-397X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2021 1 631-645 |
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10.1080/21623945.2021.1990480 doi (DE-627)DOAJ074166905 (DE-599)DOAJ55f47f5971ea489b9fdb752081c1730e DE-627 ger DE-627 rakwb eng RC648-665 QH573-671 QP1-981 Tomás Suchý verfasserin aut Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. adipocytes cas9 crispr/cas adipocyte function 3t3-l1 genetic knock-out Diseases of the endocrine glands. Clinical endocrinology Cytology Physiology Isabell Kaczmarek verfasserin aut Tomislav Maricic verfasserin aut Christian Zieschang verfasserin aut Torsten Schöneberg verfasserin aut Doreen Thor verfasserin aut Ines Liebscher verfasserin aut In Adipocyte Taylor & Francis Group, 2019 10(2021), 1, Seite 631-645 (DE-627)726123374 (DE-600)2681769-X 2162397X nnns volume:10 year:2021 number:1 pages:631-645 https://doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/article/55f47f5971ea489b9fdb752081c1730e kostenfrei http://dx.doi.org/10.1080/21623945.2021.1990480 kostenfrei https://doaj.org/toc/2162-3945 Journal toc kostenfrei https://doaj.org/toc/2162-397X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2021 1 631-645 |
| language |
English |
| source |
In Adipocyte 10(2021), 1, Seite 631-645 volume:10 year:2021 number:1 pages:631-645 |
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Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines |
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Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. |
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Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. |
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Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out. |
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The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. 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