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Hum Genomics. 2019 Feb 15;13(1):10. doi: 10.1186/s40246-019-0192-8.

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

Author information

1
Department of Biology, Miami University, Oxford, OH, 45056, USA.
2
Present Address: Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
3
Nuclear Medicine Department, University of Cincinnati Medical Center, 234 Goodman Street, Cincinnati, OH, 45219, USA.
4
Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH, 43205, USA.
5
Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Sagol School of Neurosciences, Tel Aviv University, 69978, Tel Aviv, Israel.
6
Department of Animal Sciences, Texas A&M University, College Station, TX, 77843-2471, USA.
7
Present Address: Emory Children's Center, Room 410, 2015 Uppergate Drive, Atlanta, GA, 30322, USA.
8
Department of Ophthalmology, Tufts University School of Medicine, Boston, MA, 02111, USA.
9
Department of Cell Biology and Human Anatomy, University of California, Davis One Shields Avenue, Davis, CA, 95616, USA.
10
Department of Biology, Miami University, Oxford, OH, 45056, USA. robinsm5@miamioh.edu.

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.

KEYWORDS:

Cre recombinase; Lens development; Transgenic mice

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