B25

acta2-GCaMP-ER Strain Information Sheet

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Strain Name:

Common Name: acta2-GCaMP-ER

CHROMusTM designation and lines available: R24:B25

Jackson Stock number: NA

Development: The green fluorescent calcium indicator GCaMP-ER2, an endoplasmic reticulum targeting calcium indicator with low calcium affinity and high dynamic range was developed and inserted at the ATG site of the acta2 gene in the BAC RP23-370F21 (CHORI) through homologous recombination. The resulting recombinant BAC was injected into the male pronucleus of fertilized oocytes which were then implanted into pseudo pregnant females. Resulting offspring were screened for the presence of the transgene (founders). Colonies were established from each founder and tested for expression.

Transgenic Numbers:

# pups born # founders # expressors
15 6 5

Description: The genetically encoded calcium indicator GCaMP-ER2 is expressed under control of the acta2 promoter, directing expression to smooth muscle cells including those in blood vessels, airways of the lung and gut.  Expression may be present in other smooth muscle containing tissues which have not been examined. GCaMP-ER2 is designed to respond to calcium levels in the endoplasmic reticulum. When calcium increases, a conformation change occurs resulting in an increase in fluorescence. When calcium decreases, fluorescence decreases. This mouse is useful for examining calcium signaling in the ER of smooth muscle cells.

Phenotypic Data:

IHC and native fluorescence shows appropriate subcellular localization in smooth muscle cells.  Functional analysis of these lines is ongoing to confirm that changes in calcium concentration in the ER are detected by the GCaMP-ER2 molecule.

acta2-GCaMP-ER IHC and native fluorescence

acta2-GCaMP-ER anti-GFP immunohistochemistry and native fluorescence showing GCaMP-ER expression in smooth muscle cells in vessels of the heart.

 

Genotyping Protocol:

Primer CalmF AAG GGC GAG GAG CTG TTC A
Primer CalmR CGA TCT GCT CTT CAG TCA GTT GGT
Expected size of product: 456bp
Cycling conditions:

  1. 94oC 3 minutes
  2. Repeat 30 times
    1. 30 sec @ 94oC
    2. 30 sec @ 57oC
    3. 60 sec @ 72oC
  3. 72oC 5 minutes
  4. 10oC hold

PCR results:

GCaMP PCR for strain info

 

Terms of Use: Please contact chromus@cornell.edu for information on MTA and fees.

So as to appropriately acknowledge and credit the scientists who have produced the acta2-GCaMP-ER mouse strain, it is expected that you will include these individuals in any publication that is submitted prior to the initial publication of this line from the Kotlikoff laboratory.

Acknowledgement: Please inform us of any publications resulting from the use of this mouse and use the following statement to acknowledge their development:

The mouse strain acta2-GCaMP-ER was developed by CHROMusTM which is supported by the National Heart Lung Blood Institute of the National Institute of Health under award number R24HL120847.

 

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