STOCK Rho^tm1Jlem Tg(Rho*)2PRieke/J

Stock number: 027645
Product name: 2P
Research areas: Cell Biology Research, Sensorineural Research

Description

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.

Detailed description

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.

Development

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.

Allele

Symbol: Rho^tm1Jlem
Name: targeted mutation 1, Janis Lem
MGI accession ID: MGI:2680822
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: mrho^-; Opsin -; rhodopsin-
Marker symbol: Rho
Marker name: rhodopsin
Marker synonyms: CSNBAD1; L opsin; Long Wavelength Sensitive opsin; LWS opsin; MGC:21585; MGC:25387; Noerg1; Opn2; Ops; opsin 2; Red Opsin; Rod Opsin; RP4
Chromosome: 6
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

Symbol: Tg(Rho*)2PRieke
Name: transgene insertion 2P, Fred Rieke
MGI accession ID: MGI:5640778
Generation method: Transgenic
Attributes: Inserted expressed sequence
Synonyms: 2P; S334/S338/CSM
Marker symbol: Tg(Rho*)2PRieke
Marker name: transgene insertion 2P, Fred Rieke
Chromosome: UN
Strain of origin: (C57BL/6J x DBA/2J)F1
Expressed genes: Rho,rhodopsin,mouse
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.