A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed...
Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Xu, Jian [verfasserIn] Zhu, Yongling

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2018

Schlagwörter:	Cre LoxP Lox2272 FLEX DIO

Anmerkung:	© The Author(s). 2018

Übergeordnetes Werk:	Enthalten in: BMC biotechnology - London : BioMed Central, 2001, 18(2018), 1 vom: 31. Aug.
Übergeordnetes Werk:	volume:18 ; year:2018 ; number:1 ; day:31 ; month:08

Links:	Volltext

DOI / URN:	10.1186/s12896-018-0462-x

Katalog-ID:	SPR028469437

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520			\|a Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants.
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912			\|a GBV_ILN_105
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912			\|a GBV_ILN_151
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912			\|a GBV_ILN_206
912			\|a GBV_ILN_213
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912			\|a GBV_ILN_4307
912			\|a GBV_ILN_4313
912			\|a GBV_ILN_4322
912			\|a GBV_ILN_4323
912			\|a GBV_ILN_4324
912			\|a GBV_ILN_4325
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allfields	10.1186/s12896-018-0462-x doi (DE-627)SPR028469437 (SPR)s12896-018-0462-x-e DE-627 ger DE-627 rakwb eng Xu, Jian verfasserin aut A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. Cre (dpeaa)DE-He213 LoxP (dpeaa)DE-He213 Lox2272 (dpeaa)DE-He213 FLEX (dpeaa)DE-He213 DIO (dpeaa)DE-He213 Zhu, Yongling (orcid)0000-0002-2527-402X aut Enthalten in BMC biotechnology London : BioMed Central, 2001 18(2018), 1 vom: 31. Aug. (DE-627)332164837 (DE-600)2052746-9 1472-6750 nnns volume:18 year:2018 number:1 day:31 month:08 https://dx.doi.org/10.1186/s12896-018-0462-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2190 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 18 2018 1 31 08
spelling	10.1186/s12896-018-0462-x doi (DE-627)SPR028469437 (SPR)s12896-018-0462-x-e DE-627 ger DE-627 rakwb eng Xu, Jian verfasserin aut A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. Cre (dpeaa)DE-He213 LoxP (dpeaa)DE-He213 Lox2272 (dpeaa)DE-He213 FLEX (dpeaa)DE-He213 DIO (dpeaa)DE-He213 Zhu, Yongling (orcid)0000-0002-2527-402X aut Enthalten in BMC biotechnology London : BioMed Central, 2001 18(2018), 1 vom: 31. Aug. (DE-627)332164837 (DE-600)2052746-9 1472-6750 nnns volume:18 year:2018 number:1 day:31 month:08 https://dx.doi.org/10.1186/s12896-018-0462-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2190 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 18 2018 1 31 08
allfields_unstemmed	10.1186/s12896-018-0462-x doi (DE-627)SPR028469437 (SPR)s12896-018-0462-x-e DE-627 ger DE-627 rakwb eng Xu, Jian verfasserin aut A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. Cre (dpeaa)DE-He213 LoxP (dpeaa)DE-He213 Lox2272 (dpeaa)DE-He213 FLEX (dpeaa)DE-He213 DIO (dpeaa)DE-He213 Zhu, Yongling (orcid)0000-0002-2527-402X aut Enthalten in BMC biotechnology London : BioMed Central, 2001 18(2018), 1 vom: 31. Aug. (DE-627)332164837 (DE-600)2052746-9 1472-6750 nnns volume:18 year:2018 number:1 day:31 month:08 https://dx.doi.org/10.1186/s12896-018-0462-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2190 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 18 2018 1 31 08
allfieldsGer	10.1186/s12896-018-0462-x doi (DE-627)SPR028469437 (SPR)s12896-018-0462-x-e DE-627 ger DE-627 rakwb eng Xu, Jian verfasserin aut A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. Cre (dpeaa)DE-He213 LoxP (dpeaa)DE-He213 Lox2272 (dpeaa)DE-He213 FLEX (dpeaa)DE-He213 DIO (dpeaa)DE-He213 Zhu, Yongling (orcid)0000-0002-2527-402X aut Enthalten in BMC biotechnology London : BioMed Central, 2001 18(2018), 1 vom: 31. Aug. (DE-627)332164837 (DE-600)2052746-9 1472-6750 nnns volume:18 year:2018 number:1 day:31 month:08 https://dx.doi.org/10.1186/s12896-018-0462-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2190 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 18 2018 1 31 08
allfieldsSound	10.1186/s12896-018-0462-x doi (DE-627)SPR028469437 (SPR)s12896-018-0462-x-e DE-627 ger DE-627 rakwb eng Xu, Jian verfasserin aut A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. Cre (dpeaa)DE-He213 LoxP (dpeaa)DE-He213 Lox2272 (dpeaa)DE-He213 FLEX (dpeaa)DE-He213 DIO (dpeaa)DE-He213 Zhu, Yongling (orcid)0000-0002-2527-402X aut Enthalten in BMC biotechnology London : BioMed Central, 2001 18(2018), 1 vom: 31. Aug. (DE-627)332164837 (DE-600)2052746-9 1472-6750 nnns volume:18 year:2018 number:1 day:31 month:08 https://dx.doi.org/10.1186/s12896-018-0462-x kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2119 GBV_ILN_2190 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 18 2018 1 31 08
language	English
source	Enthalten in BMC biotechnology 18(2018), 1 vom: 31. Aug. volume:18 year:2018 number:1 day:31 month:08
sourceStr	Enthalten in BMC biotechnology 18(2018), 1 vom: 31. Aug. volume:18 year:2018 number:1 day:31 month:08
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Cre LoxP Lox2272 FLEX DIO
isfreeaccess_bool	true
container_title	BMC biotechnology
authorswithroles_txt_mv	Xu, Jian @@aut@@ Zhu, Yongling @@aut@@
publishDateDaySort_date	2018-08-31T00:00:00Z
hierarchy_top_id	332164837
id	SPR028469437
language_de	englisch
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author	Xu, Jian
spellingShingle	Xu, Jian misc Cre misc LoxP misc Lox2272 misc FLEX misc DIO A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid
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topic_title	A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid Cre (dpeaa)DE-He213 LoxP (dpeaa)DE-He213 Lox2272 (dpeaa)DE-He213 FLEX (dpeaa)DE-He213 DIO (dpeaa)DE-He213
topic	misc Cre misc LoxP misc Lox2272 misc FLEX misc DIO
topic_unstemmed	misc Cre misc LoxP misc Lox2272 misc FLEX misc DIO
topic_browse	misc Cre misc LoxP misc Lox2272 misc FLEX misc DIO
format_facet	Elektronische Aufsätze Aufsätze Elektronische Ressource
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title	A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid
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title_full	A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid
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journal	BMC biotechnology
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author_browse	Xu, Jian Zhu, Yongling
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author-letter	Xu, Jian
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title_sort	rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid
title_auth	A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid
abstract	Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. © The Author(s). 2018
abstractGer	Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. © The Author(s). 2018
abstract_unstemmed	Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants. © The Author(s). 2018
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title_short	A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid
url	https://dx.doi.org/10.1186/s12896-018-0462-x
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up_date	2024-07-03T19:37:33.365Z
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A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid

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