C57BL/6J-Tg(CAG-ARL13B/mCherry/GECO1.2)1Clph/J

Stock number: 030613
Product name: hArl13b-mCherry-GECO1.2^tg
Research areas: Neurobiology Research, Research Tools

Description

Arl13b-mCherry-GECO1.2^tg transgenic mice express a ratiometric calcium indicator in all primary cilia that is useful for in vivo/in vitro assessment of Ca²⁺ signaling.

Detailed description

Arl13b-mCherry-GECO1.2^tg transgenic mice express a ratiometric calcium indicator in all primary cilia that is useful for in vivo/in vitro assessment of Ca²⁺ signaling. Evaluation of the GECO1.2 (green) and mCherry (red) fluorescence ratio (F_GECO1.2/F_mCherry) reduces movement-related artifacts.

Expression of the transgene is found in all primary cilia of the inner ear sensory epithelium, including kinocilia of both inner hair cells and outer hair cells of the organ of Corti. Scanning electron microscopy between embryonic day 18 (E18) and postnatal day 3 (P3) has confirmed normal development of stereocilia bundles in transgenic animals.

Deflection of the stereocilia bundle results in a rapid and robust increase in F_GECO1.2 within the bundle, while F_mCherry remains largely unchanged. Inner hair cell bundle deflection increases the ratio approximately 3-fold, with recovery to the resting value within 2.5s after the application of laminar flow. The slow dissociation rate of Ca²⁺ from GECO1.2 (τ approximately 600 ms) provides a valuable means of distinguishing true changes in Ca²⁺ concentration from movement-related artifacts. In contrast, kinocilia deflected by pressure steps over a range of developmental stages (E14 to P3) exhibit no detectable increase in ciliary Ca²⁺ concentration.

This model has also been used to measure cilia bending in cultured medullary collecting duct cells of the kidney, and primary cilia of the embryonic node during development.

Homozygous mice are viable and fertile, and show no organ morphology/orientation defects that would suggest cilia defects. Heterotaxy has not been observed. Breeding animals are reported to show normal litter sizes (6-8 pups on C57BL/6 background).

Development

The CAG promoter drives expression of full-length human ARL13B (ADP-ribosylation factor-like 13B), mCherry, and GECO1.2. The GECO1.2 component is an improved green fluorescent GECI (genetically encoded calcium indicator) with an apparent K_d of 450 nm. The transgene was introduced to C57BL/6J embryos and maintained on a C57BL/6J genetic background by the donating laboratory. A single copy of the transgene integrated in a non-coding region of mouse Chromosome 1 (position 174,611,546).

Allele

Symbol: Tg(CAG-ARL13B/mCherry/GECO1.2)1Clph
Name: transgene insertion 1, David E Clapham
MGI accession ID: MGI:5898420
Generation method: Transgenic
Attributes: Reporter; Humanized sequence
Synonyms: Arl13b-mCHerry-GECO1.2^tg
Marker symbol: Tg(CAG-ARL13B/mCherry/GECO1.2)1Clph
Marker name: transgene insertion 1, David E Clapham
Chromosome: 1
Strain of origin: C57BL/6J
Expressed genes: ARL13B,ADP ribosylation factor like GTPase 13B,human; RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: Expression of the transgene is found in all primary cilia of the inner ear sensory epithelium, including kinocilia of both inner hair cells and outer hair cells of the organ of Corti.
Molecular note: The CAG promoter drives expression of full-length human ARL13B (ADP-ribosylation factor-like 13B)fused to mCherry and GECO1.2. The GECO1.2 component is an improved green fluorescent GECI (genetically encoded calcium indicator) with an apparent K>d< of 450 nm. A single copy of the transgene integrated in a non-coding region of mouse Chromosome 1 (position 174,611,546).