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

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

doi:10.1186/s40246-019-0192-8

Cited by 25 publications

(19 citation statements)

References 55 publications

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“…Pairing these techniques with CAV and intersectional methods enables a wide range of combinations, allowing substantial versatility and creativity in experimental designs. It should be noted that, regardless of the vector used, long-term overexpression of Cre recombinase can be toxic (Silver and Livingston, 2001;Braz et al, 2002;Whitsett and Perl, 2006;Gong et al, 2007;Harno et al, 2013;Janbandhu et al, 2014;Lam et al, 2019), and therefore, precautions are needed when Cre is used in functional assays.…”

Section: Applicationsmentioning

confidence: 99%

Canine Adenovirus 2: A Natural Choice for Brain Circuit Dissection

Lavoie

Liu

2020

Front. Mol. Neurosci.

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Canine adenovirus-2 (CAV) is a canine pathogen that has been used in a variety of applications, from vaccines against more infectious strains of CAV to treatments for neurological disorders. With recent engineering, CAV has become a natural choice for neuroscientists dissecting the connectivity and function of brain circuits. Specifically, as a reliable genetic vector with minimal immunogenic and cytotoxic reactivity, CAV has been used for the retrograde transduction of various types of projection neurons. Consequently, CAV is particularly useful when studying the anatomy and functions of long-range projections. Moreover, combining CAV with conditional expression and transsynaptic tracing results in the ability to study circuits with cell-and/or projectiontype specificity. Lastly, with the well-documented knowledge of viral transduction, new innovations have been developed to increase the transduction efficiency of CAV and circumvent its tropism, expanding the potential of CAV for circuit analysis.

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Section: Applicationsmentioning

confidence: 99%

Canine Adenovirus 2: A Natural Choice for Brain Circuit Dissection

Lavoie

Liu

2020

Front. Mol. Neurosci.

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“…Our results indicate that Gclc appears to be required for normal eye and lens development. However, Le-Cre transgenic mice can display a microphthalmia phenotype, regardless of floxed alleles [36–39]. Therefore, the extent to which hemizygosity for the Le-Cre causes microphthalmia in our B6/FVB mixed background mice was determined (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Given that the Le-Cre transgene utilizes the Pax P0 promoter, others have postulated that it may deplete cofactors required for endogenous Pax6 expression [37]. However, a recent study was unable to confirm this hypothesis [36]. Instead, it was found that the microphthalmia phenotype (in Le-Cre homozygous mice) was caused by changes in the expression of genes involved in the negative regulation of cell proliferation and cell growth, and positive regulation of apoptosis pathways.…”

Section: Discussionmentioning

confidence: 99%

“…By the time of weaning (P21), Gclc KO eyes have small vacuolated lens, severe retinal cellular layer expansion and in folding, hypoplasia of the iris, and corneal epithelial cell layers that are hyperproliferated and poorly differentiated. However, mice that have at least one Le-Cre transgene allele may develop microphthalmia regardless of any floxed alleles [36–39]. For example, mice homozygous for the Le-Cre transgene display high penetrance microphthalmia in a strain-dependent manner [38], whereas mice hemizygous for Le-Cre transgene display much lower penetrance in a similarly strain-dependent manner [36, 37].…”

Section: Discussionmentioning

confidence: 99%

“…However, mice that have at least one Le-Cre transgene allele may develop microphthalmia regardless of any floxed alleles [36–39]. For example, mice homozygous for the Le-Cre transgene display high penetrance microphthalmia in a strain-dependent manner [38], whereas mice hemizygous for Le-Cre transgene display much lower penetrance in a similarly strain-dependent manner [36, 37]. FVB/N inbred mice served as the original background for the Le-Cre transgene [28] and Le-Cre hemizygous transgenic mice maintained on this background exhibit normal eye development.…”

Section: Discussionmentioning

confidence: 99%

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Gclc deletion in surface-ectoderm tissues induces microphthalmia

Thompson

Chen

Philippe

et al. 2019

Preprint

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Glutamate cysteine ligase catalytic subunit (Gclc) is the catalytic subunit for the glutamate-cysteine ligase (Gcl) enzyme. Gcl catalyzes the rate limiting step in glutathione (GSH) synthesis. Gclc is highly expressed in the developing eye. To define the regulatory role of Gclc in eye development, we developed a novel, Le-Cre transgene-driven, Gclc knockout mouse model. Gclc f/f /Le-Cre Tg/mice present with deformation of the retina, cornea, iris, and lens, consistent with a microphthalmia phenotype. Controlling for the microphthalmia phenotype of Gclc wt/wt /Le-Cre Tg/mice revealed that Gclc f/f /Le-Cre Tg/mice have a more severe microphthalmia phenotype. Thus, the loss of Gclc expression exacerbates the microphthalmia phenotype in Le-Cre mice.Gclc f/f /Le-Cre Tg/eyes present with reduced retinal and lens epithelium proliferation and increased lens cell death. Imaging mass spectrometry of ocular tissues revealed changes in the intensity and distribution of several lipid species and proteins in the retina and corneas of Gclc f/f /Le-Cre Tg/eyes. Lastly, using splice-blocking morpholinos and CRISPR/Cas9, we created two gclc knockdown zebrafish models, both of which display a microphthalmia phenotype. Combined, the mouse and zebrafish results indicate that, in chordates, Gclc has a conserved role in regulating eye development. In summary, these novel animal models are useful tools for elucidating the mechanisms involved in microphthalmia development.≈ 11% of the cases [1]. Currently, there is no cure for the severe loss of vision associated with microphthalmia. While surgical treatments exist, they only address the cosmetic abnormalities associated with microphthalmia [15]. However, recent research demonstrating the efficacy of nonsense suppression (inhibiting the effect of a nonsense mutation by pharmacologically increasing the likelihood of a near cognate aminoacyl-tRNA substitution) to rescue lens development when administered postnatally in a Pax6-deficient mouse model has provided hope that treatments for microphthalmia can be developed [16]. Clearly, a more complete understanding of the mechanisms regulating lens development should accelerate the discovery of preventative strategies and/or novel therapeutic treatments for microphthalmia. Glutathione (GSH), an intracellular antioxidant, is highly abundant in the eye, with concentrations of 20mM or more occurring in the lens cortex and epithelium [17, 18]. It is important for physiological detoxification of electrophiles and oxidants in the lens cortex and epithelium [19]. Through these functions, GSH maintains lens clarity and, as such, a decline in GSH levels is associated with cataract development [20, 21]. GSH biosynthesis involves a two-step enzymatic process [22] involving glutamate-cysteine ligase (GCL) followed by glutathione synthase (GSS). GCL, the rate limiting step in GSH synthesis, comprises a catalytic subunit (GCLC) and a modifier subunit (GCLM). Impaired GCLC or GCLM function limits GSH synthesis and reduces intracellular GSH concentrations. Th...

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Drebrin, an actin‐binding protein, is required for lens morphogenesis and growth

Karnam

Maddala

Stiber

et al. 2021

Developmental Dynamics

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Background: Lens morphogenesis, architecture, and clarity are known to be critically dependent on actin cytoskeleton organization and cell adhesive interactions. There is limited knowledge, however regarding the identity and role of key proteins regulating actin cytoskeletal organization in the lens. This study investigated the role of drebrin, a developmentally regulated actinbinding protein, in mouse lens development by generating and characterizing a conditional knockout (cKO) mouse model using the Cre-LoxP recombination approach. Results: Drebrin E, a splice variant of DBN1 is a predominant isoform expressed in the mouse lens and exhibits a maturation-dependent downregulation. Drebrin co-distributes with actin in both epithelium and fibers.Conditional deficiency (both haploinsufficiency and complete absence) of drebrin results in disrupted lens morphogenesis leading to cataract and microphthalmia. The drebrin cKO lens reveals a dramatic decrease in epithelial height and width, E-cadherin, and proliferation, and increased apoptotic cell death and expression of α-smooth muscle actin, together with severely impaired fiber cell organization, polarity, and cell-cell adhesion.Conclusions: This study demonstrates the requirement of drebrin in lens development and growth, with drebrin deficiency leading to impaired lens morphogenesis and microphthalmia.

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

Cited by 25 publications

References 55 publications

Canine Adenovirus 2: A Natural Choice for Brain Circuit Dissection

Canine Adenovirus 2: A Natural Choice for Brain Circuit Dissection

Gclc deletion in surface-ectoderm tissues induces microphthalmia

Drebrin, an actin‐binding protein, is required for lens morphogenesis and growth

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