RPE-specific expression of cre is useful for generating gene knockouts limited to the RPE. This facilitates study of the RPE-cell autonomous function of genes, some of which cause embryonic lethality when knocked out ubiquitously. It also facilitates generation of mouse models of human retinopathies in which the RPE harbors the primary defect. Herein, we describe generation of an RPE-specific transgenic mouse line in which maximum cre expression begins after eye development. The expressed cre is enzymatically active and nontoxic to the RPE up to at least 2 years of age.
Previous RPE cre
-expressing lines () using Dct
promoters demonstrate cre activity in the embryo.6,7
While this is advantageous for developmental studies, this embryonic deletion may cause developmental abnormalities, and therefore analysis of phenotypes in adulthood is more complicated. The BEST1-cre
transgenic mice presented herein have low-level cre
expression beginning at P10 (). At this time all retinal layers are developed. At P10, no new photoreceptors are born, although photoreceptor outer segments continue to elongate until P17.22,23
The RPE cells are proliferating at P10, as 25% more are found at P17.24
Therefore, gene knockouts within the RPE at this stage could only lead to developmental abnormalities if the effect of gene deletion is very rapid. Further, since the Trp-1
lines express cre
in several different cell types within the eye, our BEST1-cre
transgenic mice are better suited for studies involving the cell autonomous functions of the RPE.
Comparison of Transgenic Mice Expressing Cre in the RPE
Immunofluorescence of cre within the RPE of adult BEST1-cre mice displays a patchy expression pattern. The percentage of cre-positive cells is influenced by genetic background. On the pure C57BL/6 background in which these mice were generated, the percentage of cre-positive cells varies from 50% to 90%. On a mixed B6/129 background, expression is more consistently 90% of RPE cells. This mosaic expression pattern provides an internal control for studies of RPE morphology; immunolabeling with the anti-cre antibody identifies cells that will delete the gene of interest, with cre-negative neighbors serving as controls. This mosaicism may represent a slight impediment for biochemical studies, but in genetic backgrounds with 90% cre-positive RPE cells, most RPE cells will have deleted the floxed gene. In contrast, the monocarboxylate transporter 3-cre line, cre is expressed in as few as 5% to 20% of RPE cells, depending on genetic background and timing of induction.
Silencing of transgenes has been observed in some transgenic mouse lines, especially when the transgene copy number is over 100.25
line has a transgene copy number of 20, based on qPCR analysis (data not shown). Occasional BEST1-cre
mice on the pure C57BL/6 background have complete silencing of cre expression in the RPE. Progeny of silenced mice have exhibited cre
expression in 90% of RPE cells when crossed to 129 background mice. Among 23 BEST1-cre
mice on a C57BL/6 background, q-RT-PCR for cre expression in RNA isolated from RPE/choroid revealed strong correlation in the cre
expression level between the eyes of each mouse. The three silenced mice had silencing in both eyes. Thus, it is imperative in studies with our BEST1-cre
line to assess the extent of cre
expression in at least one eye from each mouse. This assessment is easily accomplished by immunofluorescence or qPCR.
A previous line using the BEST1 promoter has been generated that expresses cre in response to doxycycline induction. In addition to the complication of using oral gavage before weaning, this line may be limited by inability to detect cre-positive RPE nuclei by immunofluorescence. Lack of immunofluorescence makes it difficult to use a technique such as flat mounting to identify RPE cells that may have changes associated with gene deletion.
Functional studies described herein show that the cre in our line is not only immunoreactive but also enzymatically active, as it caused recombination between loxP sites in three floxed lines: Rosa26-lacZ
, and Heph
. The Tfam26
lines have interesting retinal degeneration phenotypes.
This transgenic mouse with RPE-limited expression of cre beginning in the late stages of postnatal eye development and a mosaic cre expression pattern should prove useful for future studies on the multifaceted functions of the RPE.