Mice carrying a transgene in which only five functional Rho (rhodopsin) phosphorylation sites (S334, T336, S338, T340, T342) are present were bred with mice lacking native Rho.
Fred Rieke, University of Washington
Rod photoreceptors generate reproducible responses to single absorbed photons in a graded and systematic manner that is dependent upon the number of phosphorylation sites in rhodopsin’s C terminus. Wildtype mouse rhodopsin has six phosphorylation sites.
In these 5P compound mutant mice, the native mouse Rho (rhodopsin) gene has been knocked out and a transgene replacing five of the functional phosphorylation sites (S334, T336, S338, T340, T342) has been introduced. The remaining phosphorylation site in the Rho transgene (S343) was inactivated via a serine to alanine mutation. An A337V mutation was introduced to confer a linear epitope for mAb3A6, that allows specific recognition of transgenic versus endogenous opsin.
Compound mutant mice in which only the S338 phosphorylation site has been replaced (1P; see Stock No. 027644) or only the S334 and S338 phosphorylation sites have been replaced (2P; see Stock No. 027645) generate single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites (e.g. the 5P mutant mice whose transgene incorporates the S334, T336, S338, T340, T342 sites) decline along smooth, reproducible time courses. The rate of recovery increases with increasing numbers of phosphorylation sites.
This strain combines a Rho knockout allele with a transgene expressing five of the six native Rho phosphorylation sites.
The Rho knockout allele was created by placing a PGK-neomycin cassette in the first coding exon of the endogenous gene. This deleted 15 bp upstream of the translation start site and the first 111 codons of the gene. The mutation was created by homologous recombination in 129S4/SvJae-derived J1 embryonic stem (ES) cells. Animals were backcrossed to C57BL/6 for at least 5 generations by the donating lab.
The transgenic portion of this strain expresses mouse Rho carrying only the S334, T336, S338, T340, and T342 phosphorylation sites. The remaining phosphorylation site in the Rho transgene (S343) was inactivated via a serine to alanine mutation. An A337V mutation was introduced to confer a linear epitope for mAb3A6, that allows specific recognition of transgenic versus endogenous opsin. The transgenic vector was introduced to B6D2F1 embryos and resultant mice were intercrossed with the targeted knockout allele to create compound mutant mice.
|Expressed Gene||Rho, rhodopsin, mouse|
|Site of Expression|
|Allele Name||targeted mutation 1, Janis Lem|
|Allele Type||Targeted (Null/Knockout)|
|Allele Synonym(s)||mrho-; Opsin -; rhodopsin-|
|Gene Symbol and Name||Rho, rhodopsin|
|Strain of Origin||129S4/SvJae|
|Molecular Note||A PGK-neo cassette was inserted into the first coding exon of the endogenous gene, deleting 15 bp upstream of the translational start site and 111 codons. Transcript was undetected in homozygous mutant mice by Northern blot and RT-PCR analyses. Western blot analysis indicated an absence of protein in whole retinal homogenates.|
|Allele Name||transgene insertion 5P, Fred Rieke|
|Allele Type||Transgenic (Inserted expressed sequence)|
|Gene Symbol and Name||Tg(Rho*)5PRieke, transgene insertion 5P, Fred Rieke|
|Promoter||Rho, rhodopsin, mouse|
|Expressed Gene||Rho, rhodopsin, mouse|
|Strain of Origin||(C57BL/6J x DBA/2J)F1|
|Molecular Note||The transgene expresses mouse Rho carrying S334, T336, S338, T340, and T342 phosphorylation sites. The remaining phosphorylation sites in the Rho transgene, S343, is inactivated via a serine to alanine mutation. An A337V mutation was introduced to confer a linear epitope for mAb3A6, that allows specific recognition of transgenic versus endogenous opsin.|
Animals homozygous or heterozygous for the Rho knockout allele are viable and fertile, as are animals hemizygous for the transgene.
When using the 5P mouse strain in a publication, please cite the originating article(s) and include JAX stock #027646 in your Materials and Methods section.