Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens | Human Genomics | Full Text

Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens | Human Genomics | Full Text

Human Genomics

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,
• Tom Glaser and
• Michael L. RobinsonEmail authorView ORCID ID profile

Human Genomics201913:10

https://doi.org/10.1186/s40246-019-0192-8

©  The Author(s). 2019

• Received: 5 December 2018
• Accepted: 22 January 2019
• Published: 15 February 2019

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-Cretransgenic 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