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. Author manuscript; available in PMC: 2013 Aug 15.
Published in final edited form as: Dev Biol. 2012 May 15;368(2):214–230. doi: 10.1016/j.ydbio.2012.05.005

Fig. 2.

Fig. 2

Characterization of the Crx>Math5-IRES-Cre and Crx>Cre conventional and BAC transgenic mice. (A) Map of conventional and BAC transgenes. In conventional transgenes, Math5-IRES-Cre pA and control Cre pA cassettes are positioned downstream of the 2.4 kb Crx promoter fragment, as Crx>Math5 Tg and Crx>Cre Tg, respectively. In BAC transgenes, equivalent cassettes were precisely inserted in BAC clone RP23-81H17, at the first ATG of the Crx gene, in exon 2, as Crx>Math5 BAC and Crx>Cre BAC. (B) Expression patterns of Crx>Math5 BAC, Crx>Cre BAC, Crx>Math5 Tg, and Crx>Cre Tg mice carrying R26floxGFP reporters. In adult and embryonic retinas for each transgene (right, far right), Crx and Cre are coextensive, suggesting faithful recapitulation of the endogenous Crx pattern. In Crx>Math5 BAC and Crx>Cre BAC retinas, reporter expression (R26floxGFP) is largely confined to the photoreceptor and bipolar precursors in the neural retina. In Crx>Math5 Tg and Crx>Cre Tg, the GFP reporter is widespread throughout the neural retina, and is present in the RPE and ciliary body (Suppl Fig. 2). (C) RT-PCR of RNA from P21 Crx>Math5 Tg tissues, P21 non-transgenic control (cnt) retinas, and E14.5 control eyes. The products show total, endogenous (endo), and transgenic (Tg) Math5, and βactin mRNA expression. Endogenous Math5 is expressed in the embyonic retina, and at low levels in the brain, but is not detected in other tissues (including the adult retina). In contrast, transgenic Math5 is strongly expressed in P21 retinas, and is restricted to eye tissue. No PCR products are detected in the absence of reverse transcriptase (− RT). (D) Taqman qRT-PCR comparing the relative abundance of Math5 and Crx transcripts in P21 Crx>Math5 BAC (BAC 60), Crx>Math5 Tg (Tg251), and control retinas. Math5 (a) and Crx (b) expression levels are normalized to Gapdh, with expression reported relative to Crx>Math5 BAC (left). Math5 expression is also reported relative to endogenous Crx levels (right), calculated using triplex competitive PCR data (k) from Crx>Math5 BAC to normalize expression levels (Suppl Fig. 3). Math5 expression in Crx>Math5 Tg retinas is 16-fold higher than Crx>Math5 BAC retinas, and 8-fold higher than endogenous Crx. Ex, exon; IRES, internal ribosome entry site; FRT, Flipase recognition target; on, optic nerve; cb, ciliary body; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar, 50 µm.