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Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.

Klimova L, Lachova J, Machon O, Sedlacek R, Kozmik Z - PLoS ONE (2013)

Bottom Line: Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited.When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup.Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
During mouse eye development, all retinal cell types are generated from the population of retina-committed progenitors originating from the neuroepithelium of the optic vesicle. Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited. Here we report generation of the mRx-Cre BAC transgenic mouse line in which the expression of Cre recombinase is controlled by regulatory sequences of the mouse Rx gene, one of the earliest determinants of retinal development. When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup. Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development. Moreover, since eye organogenesis is dependent on the inductive signals between the optic vesicle and head surface ectoderm, the inductive ability of the optic vesicle can be analyzed using mRx-Cre transgenic mice.

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Activity of Rx-Cre, MB31-Cre and mRx-Cre in the eye primordium analyzed using the ROSA26R-EYFP reporter line.(A–C) Whole-mounts showing EYFP expression (green) in the overall embryo at E10.5. (A’–C’) Coronal sections through the eye region co-stained with DAPI (blue) showing Cre activity in the retina, retinal pigmented epithelium and invaginating lens pit (dashed line) at E10.5.
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pone-0063029-g003: Activity of Rx-Cre, MB31-Cre and mRx-Cre in the eye primordium analyzed using the ROSA26R-EYFP reporter line.(A–C) Whole-mounts showing EYFP expression (green) in the overall embryo at E10.5. (A’–C’) Coronal sections through the eye region co-stained with DAPI (blue) showing Cre activity in the retina, retinal pigmented epithelium and invaginating lens pit (dashed line) at E10.5.

Mentions: In order to perform gene inactivation specifically in retinal progenitors of the optic vesicle we first reinvestigated the previously generated Rx-Cre[12]. The Rx-Cre transgene is schematically depicted in Figure 1A. To define the kinetics and pattern of the Cre recombinase activity driven by Rx-Cre, transgenic mice were bred with the ROSA26R reporter mouse strain to generate Rx-Cre; ROSA26R double transgenic animals. These mice enabled detection of the Cre activity and lineage tracing of Cre-expressing cells using X-gal staining. Upon Cre recombination, the expression of LacZ under the ROSA promoter is activated by the removal of a stop cassette [21]. The Rx-Cre; ROSA26R embryos reproducibly showed a broad area of recombination (Figure 2A,D). Already at E9.0 X-gal staining was observed both in OV and in the overall head region, also targeting head surface ectoderm (Figure 2A,A’). By E10.5 expression was detected in the neuroepithelium of the optic cup and also in the invaginating structure of the lens pit (Figure 2D’). At the early stages of vertebrate eye development, some genes are expressed in both SE and OV that interact and induce their mutual development. To study such genes it is therefore essential to perform gene inactivation in both tissues separately in order to dissect their cell autonomous functions. For this reason, Cre activity in the ectodermal compartment can be undesirable. We thus analyzed recombination in ectoderm-derived structures in more detail using the ROSA26R-EYFP reporter line [22]. The Cre-mediated recombination in ROSA26R-EYFP results in the expression of fluorescent protein EYFP which enables a better signal resolution at the single cell level than ROSA26R. The analysis of Rx-Cre; ROSA26R-EYFP embryos confirmed Cre activity in SE and invaginating lens vesicle (Figure 3A,A’). Expression of EYFP also offered the opportunity to determine the degree of mosaic recombination based on the proportion of EYFP+ and DAPI+ retinal progenitor cells. The pattern of EYFP expression revealed that at E10.5 a considerable amount of cells residing in the retina escaped to Rx-Cre-mediated deletion (Figure 3A’).


Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.

Klimova L, Lachova J, Machon O, Sedlacek R, Kozmik Z - PLoS ONE (2013)

Activity of Rx-Cre, MB31-Cre and mRx-Cre in the eye primordium analyzed using the ROSA26R-EYFP reporter line.(A–C) Whole-mounts showing EYFP expression (green) in the overall embryo at E10.5. (A’–C’) Coronal sections through the eye region co-stained with DAPI (blue) showing Cre activity in the retina, retinal pigmented epithelium and invaginating lens pit (dashed line) at E10.5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646923&req=5

pone-0063029-g003: Activity of Rx-Cre, MB31-Cre and mRx-Cre in the eye primordium analyzed using the ROSA26R-EYFP reporter line.(A–C) Whole-mounts showing EYFP expression (green) in the overall embryo at E10.5. (A’–C’) Coronal sections through the eye region co-stained with DAPI (blue) showing Cre activity in the retina, retinal pigmented epithelium and invaginating lens pit (dashed line) at E10.5.
Mentions: In order to perform gene inactivation specifically in retinal progenitors of the optic vesicle we first reinvestigated the previously generated Rx-Cre[12]. The Rx-Cre transgene is schematically depicted in Figure 1A. To define the kinetics and pattern of the Cre recombinase activity driven by Rx-Cre, transgenic mice were bred with the ROSA26R reporter mouse strain to generate Rx-Cre; ROSA26R double transgenic animals. These mice enabled detection of the Cre activity and lineage tracing of Cre-expressing cells using X-gal staining. Upon Cre recombination, the expression of LacZ under the ROSA promoter is activated by the removal of a stop cassette [21]. The Rx-Cre; ROSA26R embryos reproducibly showed a broad area of recombination (Figure 2A,D). Already at E9.0 X-gal staining was observed both in OV and in the overall head region, also targeting head surface ectoderm (Figure 2A,A’). By E10.5 expression was detected in the neuroepithelium of the optic cup and also in the invaginating structure of the lens pit (Figure 2D’). At the early stages of vertebrate eye development, some genes are expressed in both SE and OV that interact and induce their mutual development. To study such genes it is therefore essential to perform gene inactivation in both tissues separately in order to dissect their cell autonomous functions. For this reason, Cre activity in the ectodermal compartment can be undesirable. We thus analyzed recombination in ectoderm-derived structures in more detail using the ROSA26R-EYFP reporter line [22]. The Cre-mediated recombination in ROSA26R-EYFP results in the expression of fluorescent protein EYFP which enables a better signal resolution at the single cell level than ROSA26R. The analysis of Rx-Cre; ROSA26R-EYFP embryos confirmed Cre activity in SE and invaginating lens vesicle (Figure 3A,A’). Expression of EYFP also offered the opportunity to determine the degree of mosaic recombination based on the proportion of EYFP+ and DAPI+ retinal progenitor cells. The pattern of EYFP expression revealed that at E10.5 a considerable amount of cells residing in the retina escaped to Rx-Cre-mediated deletion (Figure 3A’).

Bottom Line: Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited.When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup.Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
During mouse eye development, all retinal cell types are generated from the population of retina-committed progenitors originating from the neuroepithelium of the optic vesicle. Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited. Here we report generation of the mRx-Cre BAC transgenic mouse line in which the expression of Cre recombinase is controlled by regulatory sequences of the mouse Rx gene, one of the earliest determinants of retinal development. When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup. Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development. Moreover, since eye organogenesis is dependent on the inductive signals between the optic vesicle and head surface ectoderm, the inductive ability of the optic vesicle can be analyzed using mRx-Cre transgenic mice.

Show MeSH
Related in: MedlinePlus