Mice carrying a transgene in which only two functional Rho (rhodopsin) phosphorylation sites (S334 and S338) are present were bred with mice lacking native Rho.
Fred Rieke, University of Washington
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
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Targeted (Null/Knockout) | Rho | rhodopsin |
Allele Type |
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Transgenic (Inserted expressed sequence) |
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 2P compound mutant mice, the native mouse Rho (rhodopsin) gene has been knocked out and a transgene replacing two of the functional phosphorylation sites (S334 and S338) has been introduced. The remaining phosphorylation sites in the Rho transgene (T336, T340, T342 and S343) are inactivated via serine or threonine to alanine mutations. 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) 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; see Stock No. 027646) 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 two 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 and S338 phosphorylation sites. The remaining phosphorylation sites in the Rho transgene (T336, T340, T342 and S343) are inactivated via serine or threonine to alanine mutations. 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 |
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Site of Expression |
Allele Name | targeted mutation 1, Janis Lem |
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Allele Type | Targeted (Null/Knockout) |
Allele Synonym(s) | mrho-; Opsin -; rhodopsin- |
Gene Symbol and Name | Rho, rhodopsin |
Gene Synonym(s) | |
Strain of Origin | 129S4/SvJae |
Chromosome | 6 |
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 2P, Fred Rieke |
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Allele Type | Transgenic (Inserted expressed sequence) |
Allele Synonym(s) | 2P; S334/S338/CSM |
Gene Symbol and Name | Tg(Rho*)2PRieke, transgene insertion 2P, Fred Rieke |
Gene Synonym(s) | |
Promoter | Rho, rhodopsin, mouse |
Expressed Gene | Rho, rhodopsin, mouse |
Strain of Origin | (C57BL/6J x DBA/2J)F1 |
Chromosome | UN |
Molecular Note | The transgene expresses mouse Rho carrying only the S334 and S338 phosphorylation sites. The remaining phosphorylation sites in the Rho transgene (T336, T340, T342 and S343) are inactivated via serine or threonine to alanine mutations. 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 2P mouse strain in a publication, please cite the originating article(s) and include JAX stock #027645 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Heterozygous for Rho<tm1Jlem>, Hemizygous or non carrier for Tg(Rho*)2PRieke |
Frozen Mouse Embryo | STOCK Rho<tm1Jlem> Tg(Rho*)2PRieke/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | STOCK Rho<tm1Jlem> Tg(Rho*)2PRieke/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | STOCK Rho<tm1Jlem> Tg(Rho*)2PRieke/J Frozen Embryo | $3373.50 |
Frozen Mouse Embryo | STOCK Rho<tm1Jlem> Tg(Rho*)2PRieke/J Frozen Embryo | $3373.50 |
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