STOCK Rho^tm1Jlem Tg(Rho*)TTM1Rieke/J

Stock number: 027648
Product name: TTM1
Research areas: Cell Biology Research, Sensorineural Research

Description

Mice carrying a transgene in which only the serine-associated Rho (rhodopsin) phosphorylation sites are functional were bred with mice lacking native Rho to create these TTM1 (threonine triple mutant) compound mutant animals.

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 TTM1 (threonine triple mutant) compound mutant mice, the native mouse Rho (rhodopsin) gene has been knocked out and a Rho transgene expressing three serine-associated phosphorylation sites has been introduced. Threonines 336, 340 and 342 have been converted to inactivating alanines. Serine-associated phosphorylation sites S334, S338 and S343 remain functional. An A337V mutation was introduced to confer a linear epitope for mAb3A6, that allows specific recognition of transgenic versus endogenous opsin.

Serine-only rhodopsin (also see Stock 027649) generates prolonged step-like single-photon responses that terminate abruptly and randomly. Threonine-only rhodopsin (see Stock 027647) generates single-photon responses that are only modestly slower than normal.

Comparisons of this TTM1 line and the TTM4 line (see Stock No. 027649) by the donating investigator indicate that there are some differences in the morphology of rod outer segments, and depending on the exact genetic background (i.e. if crossed with another line) one or the other may have healthier rod photoreceptors.

Development

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 transgene expresses mouse Rho in which phosphorylating threonines 336, 340 and 342 have been converted to inactivating alanines. Serine-associated phosphorylation sites (S334, S338 and S343) remain functional. 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
Marker symbol: Rho
Marker name: rhodopsin
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*)TTM1Rieke
Name: transgene insertion TTM1, Fred Rieke
MGI accession ID: MGI:5641168
Generation method: Transgenic
Attributes: Inserted expressed sequence
Marker symbol: Tg(Rho*)TTM1Rieke
Marker name: transgene insertion TTM1, 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 in which phosphorylating threonines 336, 340 and 342 have been converted to inactivating alanines. Serine-associated phosphorylation sites (S334, S338 and S343) remain functional. An A337V mutation was introduced to confer a linear epitope for mAb3A6, that allows specific recognition of transgenic versus endogenous opsin.