Tg(CAG-GFPuv/AEQ*)30Alns
Transgene Detail

Summary

• Symbol: Tg(CAG-GFPuv/AEQ*)30Alns
• Name: transgene insertion 30, Maria Teresa Alonso
• MGI ID: MGI:5800664
• Synonyms: erGAP1, erGAP1 L30, Tg(CAG-GAP1)30
• Transgene: Tg(CAG-GFPuv/AEQ*)30Alns Location: unknown
• Alliance: Tg(CAG-GFPuv/AEQ*)30Alns page

Transgene origin

• Strain of Origin: C57BL/6 or CBA

Transgene description

• Transgene Type: Transgenic (Inserted expressed sequence, Reporter)
• Mutation: Insertion
• Mutation details: Mice bearing this transgene express a fluorescent Ca²⁺ reporter protein that is targeted to the endoplasmic reticulum (ER). "GAP" is a fusion protein comprising a modified green fluorescent protein, GFPuv (GFP with amino acid substitutions Q80R, F99S, M153T, and V163A) joined by a 16-amino acid linker peptide (TATPATTPTTAPTAGT) to the Ca²⁺-sensing bioluminescent protein aequorin. To improve its performance in the ER, whose [Ca²⁺] is greater than cytosolic [Ca²⁺], its Ca²⁺ sensitivity was reduced by replacement of the acidic amino acid aspartate with alanine at three positions in the aequorin moiety (D117A, D119A, and D163A) to create "GAP1." Targeting of this ER-optimized Ca²⁺ sensor to the ER was achieved by appending the calreticulin signal peptide and the ER retention signal (KDEL) to its 5' and 3' termini, respectively, to generate "erGAP1." The construct encoding erGAP1 was cloned into the pCAGGS expression vector between the cytomegalovirus immediate early enhancer/chicken beta-actin promoter (CMV-IE/ACTB) and the rabbit beta-globin polyadenylation signal and 3'-untranslated region (3'-UTR).

  In mice of three high copy number transgenic lines (lines 11, 20 and 30) exhibiting preferential expression of erGAP1 in neural tissues, GFP fluorescence is first observed at embryonic days E12-E13 and is stable at least through 18 months. erGAP1 is strongly expressed in pyramidal CA1 and CA2 neurons and the dentate gyrus of the hippocampus, in the cortex, in both granule neurons and Purkinje cells of the cerebellum, and in dorsal root ganglion (DRG) neurons and the spinal cord. In all brain regions examined, erGAP1 is correctly targeted to the ER without evidence of aggregation. Its [Ca²⁺] sensor function in hippocampal and spinal sections and in individual neurons of various types isolated from these mice was demonstrated by reproducible, rapid, reversible changes in the F470/F405 fluorescence intensity in response to glutamate and caffeine at different concentrations. (J:206829)

Find Mice (IMSR)

• Mouse strains and cell lines available from the International Mouse Strain Resource (IMSR)
• Carrying this Mutation: Mouse Strains: 1 strain available Cell Lines: 0 lines available

Notes

"erGAP1 cDNA was cloned into the pCAG-GS expression vector and injected into B6CBAF2 [(C57BL/6 x CBA)F2] fertilized eggs." (J:206829)

References

• Original: J:206829 Rodriguez-Garcia A, et al., GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles. Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2584-9
• All: 1 reference(s)