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mice (Le et al. 2008), we used a 3.0-kb human VMD2 promoter to direct the expression of the tetracycline inducible transactivator rtTA, which in turn, should drive the expression of the tetracycline-responsive element (TRE) controlled cre gene in the presence of doxycycline. The VMD2-cre transgenic mice were generated by coinjection of linearized and purified VMD2-rtTA and TRE-cre DNA (see Le et al. for detail 2008). Analyzing segregation patterns of all transgenic founders and their progenies by PCR indicated that both transgenes were co-integrated to a single chromosome in all transgenic animals (data not shown). All germline-transmitted mice were normal in size, morphology, and behavior, and were characterized further.
24.3.2 Localization of Cre Function in Transgenic Mice
To localize Cre expression, all cre-positive mice were bred with R26R mice. R26R mice were genetically modified so that lacZ gene could be activated after Cremediated excision of a loxP-flanked transcriptional ‘STOP’ DNA segment (Soriano 1999). β-Galactosidase staining from double transgenic F1 VMD2-cre/R26R mice was used to localize Cre activity. Of the ten VMD2-cre transgenic lines identified in our initial screening for Cre function, four lines were selected for further characterization, based on the levels and locations of Cre expression. Two lines demonstrated homogeneous Cre activity in the RPE. Figure 24.1b, d shows Cre activated β-galactosidase reporter was expressed in the RPE of a VMD2-cre transgenic line (Le et al. 2008). Two lines demonstrated predominant Cre activity in Müller cells. Figure 24.1a, c shows Cre-activated β-galactosidase reporter was expressed in the Müller cells of another VMD2-cre mouse line (Ueki et al. 2009).
The transgenic line with Cre activity in Müller cells (Fig. 24.1c) also demonstrated some Cre activity in photoreceptor inner segment and what appeared to be bipolar and horizontal cells (Ueki et al. 2009). However, we were not able to detect Cre protein with Western blot and immunohistochemistry. To determine the initiation of Cre activity, this line of VMD2-cre mice were bred with floxed gp130 (gp130 f f ) mice (Betz et al. 1998). In VMD2-cre+/gp130 f f mice, Cre-mediated recombination occurred as early as embryonic day 15 (Fig. 24.2). Therefore, it is most likely that Cre was expressed through a brief transcription from the VMD2 promoter during embryonic development in retinal progenitor cells. The efficiency of Cre-mediated recombination at gp130 locus was 52% in Müller cells in this transgenic line (Ueki et al. 2009), a reasonable frequency for cell-specific gene knockout studies. Despite our efforts to generate mice with inducible Cre expression, this mouse line demonstrated limited inducibility in respect to Cre expression.
24.4 Discussion
In this study, we used a 3.0-kb human VMD2 promoter to direct tetracyclineinducible system controlled Cre expression in transgenic mice. Most transgenic lines demonstrated Cre function in the RPE. The fact that approximately half of the
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Fig. 24.1 Localization and functional analysis of Cre expression with β-galactosidase staining in F1 double transgenic VMD2-cre/R26R mice. a, c: Representative result of β-galactosidase staining (dark staining) in retinal sections of a VMD2-cre mouse line showing identifiable staining in retinal Müller cells (Arrows in c) and unidentifiable staining in neurons in inner nuclear layer (arrowheads). b, d: Representative result of β-galactosidase staining in the RPE (arrows in d) of a VMD2-cre mouse line. Scale bar equals to 100 μm. Outer nuclear layer (ONL) and inner nuclear layer (INL) are labeled. Cre function was localized to photoreceptor inner segment, Müller cells, and unknown INL neurons in one VMD2-cre mouse line (a, c) and the RPE in another VMD2-cre mouse line (b, d)
transgenic lines demonstrated Cre activated reporter expression in retinal Müller cells (data not shown) suggest that this unanticipated Cre expression is likely an intrinsic characteristic of the VMD2 promoter. However, more studies are needed to confirm this point. As the 3.0-kb VMD2 promoter used in this study may not contain all control elements necessary for identical expression patterns of the endogenous gene and genetic elements in our two gene system may affect transcription, we can not completely rule out that the unanticipated Cre function in Müller cells may be caused by the interaction between the tetracycline-inducible gene expression system and the VMD2 promoter during transcription. Although Esumi et al. demonstrated that a 585-bp VMD2 promoter was capable of targeting β-galactosidase reporter expression to the RPE exclusively in transgenic mice (Esumi et al. 2004), we need to point out that there is a fundamental difference between a VMD2 promoter directly controlled reporter expression and our study. In their study, the β-galactosidase activity reflects the cellular location of constitutive VMD2 promoter activity and any transient activity during embryonic development may not be detected in the assay. Since Cre-mediated excision is usually permanent in vivo, a transient Cre
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Fig. 24.2 PCR analysis of Cre-mediated recombination in the retina of a VMD2-cre +/gp130ff mouse using retinal and tail DNA. A VMD2-cre –/gp130ff mouse was used as a control. By embryonic day 15, Cre function was detected in the retina of VMD2-cre +/gp130ff mice
expression at any developmental stage will permit the expression of Cre-activated β-galactosidase reporter permanently, under the control of a generalized promoter (ROSA26) (Fig. 24.1). Therefore, the effect of transient VMD2 promoter activity is likely ‘amplified’ in our study. As Cre-mediated recombination was detectable in the retina at embryonic day 15 in cells that ultimately became Müller cells (Ueki et al. 2009), it is reasonable to conclude that VMD2 promoter is transcriptionally active in progenitors of Müller cells. Due to a lack of information about the expression pattern of β-galactosidase reporter under the direct control of 3.0-kb VMD2 promoter in transgenic mice, we are not in a position to conclude if the 3.0-kb VMD2 promoter confers transcriptional activity outside the RPE. Since we were not able to detect Cre expression in postnatal retina by Western blots or immunohistochemistry, it is very unlikely that the 3.0-kb VMD2 promoter is active in mature Müller cells. At this time, it is unclear if the VMD2 protein is made in non-RPE lineage during development. If so, the physiological significance of VMD2 protein in these cells remains to be determined. Nevertheless, our study indicates a possibility that transcription of VMD2 gene occurs in non-RPE lineage during retinal development.
In summary, we use the VMD2 promoter to generate inducible RPE-specific Cre mice for conditional gene activation and inactivation. In the process, we obtained transgenic mouse line with additional utility, i.e. transgenic mice expressing Cre in Müller cells with a reasonable efficiency (Ueki et al. 2009). Since the retinal integrity is essential to the usefulness of retinal cell-specific cre mice, classical parameters used in characterizing this type of mice (Le et al. 2004; Le et al. 2006), we also investigated the retinal integrity of the two VMD2-cre mouse lines (discussed here) for up to 10 months. We did not detect any abnormality in the retinal integrity in these mice, as documented in our original descriptions (Le et al. 2008; Ueki et al. 2009). The 10-month window provides crucial time required for most conditional gene knockout studies. Therefore, these two VMD2-cre mouse lines will
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be useful tools for dissecting cellular mechanisms of retinal diseases, particularly for age-related macular degeneration and diabetic retinopathy, two leading causes of blindness with high relevance to gene expression in the RPE and Müller cells.
Acknowledgments We thank W. Zheng and Y. W. Le for technical assistance and Drs. N. Esumi and D. Zack for providing human VMD2 promoter DNA. This study was supported by NIH grants RR17703, EY16459, and EY12190, ADA grant 1-06-RA-76, AHAF grant M2008-059, FFB grant BR-CMM-0808-0453-UOK and unrestricted grants from Hope for Vision and Research to Prevent Blindness.
References
Betz UA, Bloch W, van den Broek M et al (1998) Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects. J Exp Med 188:1955–1965
Esumi N, Oshima Y, Li Y et al (2004) Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation. J Biol Chem 279:19064–19073
Le Y, Ash JD, Al-Ubaidi MR et al (2004) Targeted expression of Cre recombinase to cone photoreceptors in transgenic mice. Mol Vis 10:1011–1018
Le YZ, Zheng W, Rao PC et al (2008) Inducible expression of cre recombinase in the retinal pigmented epithelium. Invest Ophthalmol Vis Sci 49:1248–1253
Le Y, Zheng L, Zheng W et al (2006) Mouse opsin promoter controlled expression of Cre recombinase in transgenic mice. Mol Vis 12:389–398
Marquardt A, Stohr H, Passmore LA et al (1998) Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best’s disease). Hum Mol Genet 7:1517–1525
Petrukhin K, Koisti MJ, Bakall B et al (1998) Identification of the gene responsible for best macular dystrophy. Nat Genet 19:241–247
Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71
Tsunenari T, Sun H, Williams J et al (2003) Structure-function analysis of the bestrophin family of anion channels. J Biol Chem 278:41114–41125
Ueki Y, Ash JD, Zhu M, Zheng L, Le Y-Z (2009) Expression of Cre recombinase in the retinal Müller cells. Vis Res 49:615–621
Chapter 25
Microarray Analysis of Hyperoxia Stressed
Mouse Retina: Differential Gene Expression
in the Inferior and Superior Region
Yuan Zhu, Riccardo Natoli, Krisztina Valter, and Jonathan Stone
Abstract
Aim: Hyperoxia-induced photoreceptor degeneration occurs preferentially in the inferior retina of C57BL/6J mice. This study investigates differential gene expression in the inferior and superior retina of C57BL/6J mouse, before and after hyperoxic stress.
Methods: At the age of P (postnatal day) 83–90, mice were placed in constant normoxia or hyperoxia (75% O2) for 2 weeks. Retinas from control and exposed mice were removed and RNA was extracted from superior and inferior regions. The RNA from 2 animals (1 male and 1 female) at each condition was extracted, purified and hybridized to an Affymetrix MouseGene 1.0 ST Array to elucidate gene expression. Experiments were run in triplicate and analysis of the expression patterns was performed using GeneSpring and Partek Genomics Suite softwares.
Results: Over 400 genes showed significant differential expression by location and treatment using 2-way ANOVA analysis. In the control material, no genes showed a differential expression greater than twofold between inferior and superior retina. After hyperoxic stress, 154 genes in the inferior and 30 genes in the superior retina showed a greater than twofold change in expression. Among those, genes such as Edn2, GFAP, Bcl3 and C1qb showed expression differences of greater than three fold between inferior and superior retina. Real time PCR was used to verify gene expression of control genes as well as genes of interest.
Conclusion: These microarray data may provide clues for identifying previously unknown factors and pathways responsible for the vulnerability of inferior retina to hyperoxic stress and for the eventual identification of therapeutic targets.
Y. Zhu (B)
School of Biology and ARC Centre of Excellence in Vision Science, The Australian National University, RSBS, Bldg 46, Biology Place, Canberra, ACT2601, Australia
e-mail: yuan.zhu@anu.edu.au
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Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_25,C Springer Science+Business Media, LLC 2010