Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens

Phuong T. Lam, Stephanie L. Padula, Thanh V. Hoang, Justin E. Poth, Lin Liu, Chun Liang, Adam S. LeFever, Lindsay M. Wallace, Ruth Ashery-Padan, Penny K. Riggs, Jordan E. Shields, Ohad Shaham, Sheldon Rowan, Nadean L Brown, Thomas M Glaser, Michael L. Robinson

Research output: Contribution to journalArticle

Abstract

BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

Original languageEnglish (US)
Number of pages1
JournalHuman genomics
Volume13
Issue number1
DOIs
StatePublished - Feb 15 2019

Fingerprint

Gene Deletion
Lenses
Transgenes
Transgenic Mice
Eye Abnormalities
Genes
Cre recombinase
Microphthalmos
Crystallins
Gene Targeting
DNA
Homozygote
Gene Expression Profiling
Reporter Genes
Pancreas
Embryonic Structures
Apoptosis

Keywords

  • Cre recombinase
  • Lens development
  • Transgenic mice

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery

Cite this

Lam, P. T., Padula, S. L., Hoang, T. V., Poth, J. E., Liu, L., Liang, C., ... Robinson, M. L. (2019). Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens. Human genomics, 13(1). https://doi.org/10.1186/s40246-019-0192-8

Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens. / Lam, Phuong T.; Padula, Stephanie L.; Hoang, Thanh V.; Poth, Justin E.; Liu, Lin; Liang, Chun; LeFever, Adam S.; Wallace, Lindsay M.; Ashery-Padan, Ruth; Riggs, Penny K.; Shields, Jordan E.; Shaham, Ohad; Rowan, Sheldon; Brown, Nadean L; Glaser, Thomas M; Robinson, Michael L.

In: Human genomics, Vol. 13, No. 1, 15.02.2019.

Research output: Contribution to journalArticle

Lam, PT, Padula, SL, Hoang, TV, Poth, JE, Liu, L, Liang, C, LeFever, AS, Wallace, LM, Ashery-Padan, R, Riggs, PK, Shields, JE, Shaham, O, Rowan, S, Brown, NL, Glaser, TM & Robinson, ML 2019, 'Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens', Human genomics, vol. 13, no. 1. https://doi.org/10.1186/s40246-019-0192-8
Lam, Phuong T. ; Padula, Stephanie L. ; Hoang, Thanh V. ; Poth, Justin E. ; Liu, Lin ; Liang, Chun ; LeFever, Adam S. ; Wallace, Lindsay M. ; Ashery-Padan, Ruth ; Riggs, Penny K. ; Shields, Jordan E. ; Shaham, Ohad ; Rowan, Sheldon ; Brown, Nadean L ; Glaser, Thomas M ; Robinson, Michael L. / Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens. In: Human genomics. 2019 ; Vol. 13, No. 1.
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AU - Lam, Phuong T.

AU - Padula, Stephanie L.

AU - Hoang, Thanh V.

AU - Poth, Justin E.

AU - Liu, Lin

AU - Liang, Chun

AU - LeFever, Adam S.

AU - Wallace, Lindsay M.

AU - Ashery-Padan, Ruth

AU - Riggs, Penny K.

AU - Shields, Jordan E.

AU - Shaham, Ohad

AU - Rowan, Sheldon

AU - Brown, Nadean L

AU - Glaser, Thomas M

AU - Robinson, Michael L.

PY - 2019/2/15

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N2 - BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

AB - BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

KW - Cre recombinase

KW - Lens development

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