VMD2-Cre-RPE transgenic mice have Tet-inducible/controllable expression of Cre recombinase directed to retinal pigmented epithelium. These mice may be useful for generating tissue specific-targeted mutants for studying gene function in retinal pigmented epithelium, especially in relation to retinal and choroidal diseases.
Yun Le, University of Oklahoma Health Sciences Center
These VMD2-Cre-RPE transgenic mice have a tetracycline (doxycycline) inducible Cre-mediated recombination system that is specific for the retinal pigmented epithelium (RPE). The tetO-PhCMV-cre transgenic construct contains cre recombinase under the control of the tetO, tetracycline-responsive regulatory element and the PVMD2-rtTA transgenic construct contains the reverse tetracycline-controlled transactivator, rtTA (Tet-On), under the control of the human BEST1, bestrophin 1, gene promoter. Mice hemizygous for the transgenic inserts are viable and fertile. The Donating Investigator expects homozygotes to be viable and fertile. Homozygous viability/fertility has not been tested (January 2019).
When crossed to a beta-galactosidase expressing reporter strain (R26R reporter mice), tetracycline (doxycycline)-inducible Cre recombinase activity was detected in retinal pigmented epithelial cells. Weak reporter expression in the optic nerve was also detected. A very slight amount of non-induced "leaky" reporter expression is detected in a few RPE cells. While induction at embryonic day 7 to 8 did not result in productive Cre recombinase activity, induction at embryonic day 9 to 10 results in detectable reporter expression in the RPE. The strongest Cre recombinase activity is obtained with induction between embryonic day 17 and postnatal day 5 in mice induced via doxycycline diet fed to mothers. Induction by direct doxycycline feeding to young mice at postnatal day 4 results in peak Cre recombinase activity levels, which corresponds to the highest level of endogenous Best1 and human BEST1 mRNA expression at about postnatal day 4. Induction of Cre recombinase is possible up to at least 2 months of age. Transgene expression in 10 month old transgenic mice that had received doxycycline treatment at weaning age does not alter RPE cell number or electroretinography (ERG) characteristics, and photoreceptor degeneration was not detected.
A transgenic construct containing cre, cre recombinase, followed by a mouse mouse metallothionein (Mt1) polyadenylation signal sequence, under the control of the tetO, tetracycline-responsive regulatory element (tetO-PhCMV-cre) and a second transgenic construct containing rtTA, reverse tetracycline-controlled transactivator under the control of the human BEST1, bestrophin 1, gene promoter (PVMD2-rtTA) were utilized in this strain. The two transgenic constructs were coinjected into fertilized FVB/N mouse eggs. Founder line 1 was subsequently established. The mice were then backcrossed occasionally to FVB wildtype mice. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize FVB/NJ oocytes (Stock No. 001800).
In 2019, 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains was performed on the rederived living colony at The Jackson Laboratory Repository. The SNP analysis revealed that this strain has unknown contributions on Chromosomes 12, 5, and 10, and is on a mixed STOCK background.
|Allele Name||transgene insertion 1, Yun-Zheng Le|
|Allele Type||Transgenic (Recombinase-expressing, Inducible, Transactivator)|
|Allele Synonym(s)||PVMD2-rtTA::tetO-PhCMVcre; VMD2-cre|
|Gene Symbol and Name||Tg(BEST1-rtTA,tetO-cre)1Yzl, transgene insertion 1, Yun-Zheng Le|
|Strain of Origin||FVB/N|
|Molecular Note||Two transgenic fragments were co-injected to generate the mouse line. One fragment contains a 3 kb human BEST1 promoter fragment driving expression of the tetracycline-inducible transactivator rtTA and the SV40 polyA sequence. The second fragment consists of the tetracycline-responsive regulatory element (tetO) and human cytomegalovirus immediate early enhancer (hCMV) controlling expression of a translationally optimized cre sequence followed by the mouse metallothionein (Mt1) polyadenylation signal. After co-injection into zygotes, 10 founders were obtained with co-integration of both fragments into a single chromosome being observed in all founders. One line demonstrating substantial activity in Muller glial cells was characterized further.|
When maintaining a live colony, hemizygous mice may be bred to wildtype siblings, or to FVB/NJ inbred mice (Stock No. 001800). The Donating Investigator expects homozygotes to be viable and fertile. Homozygous viability/fertility has not been tested (January 2019).
When using the PVMD2-rtTA::tetO-PhCMVcre, VMD2-Cre-RPE mouse strain in a publication, please cite the originating article(s) and include JAX stock #032910 in your Materials and Methods section.
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