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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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The mRx-Cre activity in the eye, forebrain and hypothalamus analyzed using the ROSA26R reporter line.Whole-mounts or sections were stained with X-gal at indicated stages to show the mRx-Cre-mediated Cre activity. (A) The X-gal+ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.
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pone-0063029-g004: The mRx-Cre activity in the eye, forebrain and hypothalamus analyzed using the ROSA26R reporter line.Whole-mounts or sections were stained with X-gal at indicated stages to show the mRx-Cre-mediated Cre activity. (A) The X-gal+ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.

Mentions: To generate mRx-Cre transgenic mouse we selected a BAC clone (RP24-82D21) containing the entire mouse Rx gene as well as 95 kb upstream of the Rx translational start site and 100 kb downstream of the locus. We employed the method of BAC recombineering [23] to insert the Cre recombinase coding sequence into the first ATG of Rx gene (Figure 1C). As the 200-kb BAC clone is supposed to carry all cis-regulatory sequences ensuring proper spatiotemporal expression, the expression pattern of Cre recombinase should imitate that of the endogenous Rx gene. The Cre-inserted BAC was used for pronuclear injection to generate mRx-Cre transgenic mice. We obtained three founders showing expression in the developing retina (not shown). One of them was chosen for further analysis using the ROSA26R reporter line. As already mentioned, endogenous Rx expression starts between E7.5 and E8.0 [14]. In agreement, we observed the mRx-Cre activity already from E8.5 in the optic sulcus, albeit with a frequent appearance of mosaic recombination (Figure 4A) of mRx-Cre; ROSA26R embryos. This slight delay can be explained by a short intermission between the Cre expression onset and recombination seen in ROSA26R. Consistent with the expression pattern of the endogenous Rx gene [14], strong X-gal staining was observed in the optic vesicles of E9.0 mRx-Cre; ROSA26R embryos (Figure 2C). Strikingly, at E10.5, the X-gal+ progeny of Cre-expressing cells contributed to all cells of the retina and the majority of retinal pigmented epithelium cells (Figure 2F’). Therefore, mRx-Cre directed Cre activity in all optic vesicle derivates, mainly to the forming retina. This almost absolute recombination rate was further confirmed in mRx-Cre; ROSA26R-EYFP eyes at E10.5 showing that virtually all retinal progenitor cells were EYFP+ (Figure 3C’). The observation that mRx-Cre targets all retinal progenitors in the early stages of eye development was further documented by uniform X-gal staining of all cellular layers of the mRx-Cre; ROSA26R adult retina (Figure 4E,E’). Importantly, no EYFP+ cells were observed in the invaginating lens pit of mRx-Cre; ROSA26R-EYFP embryos (Figure 3C’). In addition, no cells showing recombination were observed in other parts of the embryo than eye, ventral forebrain and hypothalamus. Beside strong β-galactosidase activity in OV-derived structures, a strong although mosaic activity was found in the ventral part of forebrain and in prospective hypothalamus (Figure 4B–D’). Although strong X-gal staining was observed in E15.5 hypothalamus and forebrain after whole-mount staining (Figure 4D–D’), sectioning of E15.5 and adult brains revealed that the mRx-Cre activity in the forebrain/cortex is strongly mosaic (Figure 4D’,F). In contrast, the rate of recombination in the hypothalamus appeared very high and we propose that this driver line may be used for genetic studies in the hypothalamus.


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)

The mRx-Cre activity in the eye, forebrain and hypothalamus analyzed using the ROSA26R reporter line.Whole-mounts or sections were stained with X-gal at indicated stages to show the mRx-Cre-mediated Cre activity. (A) The X-gal+ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646923&req=5

pone-0063029-g004: The mRx-Cre activity in the eye, forebrain and hypothalamus analyzed using the ROSA26R reporter line.Whole-mounts or sections were stained with X-gal at indicated stages to show the mRx-Cre-mediated Cre activity. (A) The X-gal+ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.
Mentions: To generate mRx-Cre transgenic mouse we selected a BAC clone (RP24-82D21) containing the entire mouse Rx gene as well as 95 kb upstream of the Rx translational start site and 100 kb downstream of the locus. We employed the method of BAC recombineering [23] to insert the Cre recombinase coding sequence into the first ATG of Rx gene (Figure 1C). As the 200-kb BAC clone is supposed to carry all cis-regulatory sequences ensuring proper spatiotemporal expression, the expression pattern of Cre recombinase should imitate that of the endogenous Rx gene. The Cre-inserted BAC was used for pronuclear injection to generate mRx-Cre transgenic mice. We obtained three founders showing expression in the developing retina (not shown). One of them was chosen for further analysis using the ROSA26R reporter line. As already mentioned, endogenous Rx expression starts between E7.5 and E8.0 [14]. In agreement, we observed the mRx-Cre activity already from E8.5 in the optic sulcus, albeit with a frequent appearance of mosaic recombination (Figure 4A) of mRx-Cre; ROSA26R embryos. This slight delay can be explained by a short intermission between the Cre expression onset and recombination seen in ROSA26R. Consistent with the expression pattern of the endogenous Rx gene [14], strong X-gal staining was observed in the optic vesicles of E9.0 mRx-Cre; ROSA26R embryos (Figure 2C). Strikingly, at E10.5, the X-gal+ progeny of Cre-expressing cells contributed to all cells of the retina and the majority of retinal pigmented epithelium cells (Figure 2F’). Therefore, mRx-Cre directed Cre activity in all optic vesicle derivates, mainly to the forming retina. This almost absolute recombination rate was further confirmed in mRx-Cre; ROSA26R-EYFP eyes at E10.5 showing that virtually all retinal progenitor cells were EYFP+ (Figure 3C’). The observation that mRx-Cre targets all retinal progenitors in the early stages of eye development was further documented by uniform X-gal staining of all cellular layers of the mRx-Cre; ROSA26R adult retina (Figure 4E,E’). Importantly, no EYFP+ cells were observed in the invaginating lens pit of mRx-Cre; ROSA26R-EYFP embryos (Figure 3C’). In addition, no cells showing recombination were observed in other parts of the embryo than eye, ventral forebrain and hypothalamus. Beside strong β-galactosidase activity in OV-derived structures, a strong although mosaic activity was found in the ventral part of forebrain and in prospective hypothalamus (Figure 4B–D’). Although strong X-gal staining was observed in E15.5 hypothalamus and forebrain after whole-mount staining (Figure 4D–D’), sectioning of E15.5 and adult brains revealed that the mRx-Cre activity in the forebrain/cortex is strongly mosaic (Figure 4D’,F). In contrast, the rate of recombination in the hypothalamus appeared very high and we propose that this driver line may be used for genetic studies in the hypothalamus.

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