Acta2-GCaMP-ER2 transgenic mice exhibit high fluorescence in the endoplasmic reticulum (ER) of smooth muscle cells (e.g., blood vessels, lung airways and gut) at both low and high calcium concentrations - making them useful as a fluorescent marker of smooth muscle cell ER, but not useful for detecting changes in calcium concentration.
Michael I Kotlikoff, Cornell University
Acta2-GCaMP-ER2 transgenic mice express an endoplasmic reticulum (ER)-targeted green fluorescent calcium indicator variant GCaMP-ER2 under control of the Acta2 locus promoter/enhancer regions within the BAC transgene. Acta2-GCaMP-ER2 mice from founder line B25-6 exhibit high expression levels of GCaMP-ER2 in endoplasmic reticulum of smooth muscle cells (e.g., blood vessels, lung airways and gut) - consistent with endogenous Acta2. This Acta2-GCaMP-ER2 mouse line exhibits high fluorescence in the ER at both low and high calcium concentrations - making it useful as a fluorescent marker of smooth muscle cell ER, but not useful for detecting changes in calcium concentration. Additional details described further below.
For Acta2-GCaMP-ER2 transgenic mice, the donating investigator reports that expression patterns appear to mimic the endogenous Acta2 expression (smooth muscle cells in blood vessels, lung airways and gut). Specifically, robust expression in the endoplasmic reticulum of smooth muscle cells in vessels of the heart (with the appropriate subcellular localization) was demonstrated via native fluorescence, as well as by immunohistochemistry staining. Expression in other tissues was not examined to date (July 2019). Although designed as a calcium indicator, the donating investigator reports that extensive analysis of this Acta2-GCaMP-ER2 mouse line revealed it to have high fluorescence at both low and high calcium concentrations - making it useful as a fluorescent marker of endoplasmic reticulum in smooth muscle cells, but not useful for detecting changes in calcium concentration. Additionally, it could be useful as a marker of sarcoplasmic reticulum in vascular muscle cells.
Mice hemizygous for the Acta2-GCaMP-ER2 transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. To date (July 2019), it has not been attempted to make this strain homozygous.
This mouse model is available by way of a collaborative effort between Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus) and The Jackson Laboratory.
Acta2-GCaMP-ER2 transgenic mice (also called Acta2-GCaMP-ER or Acta2BAC-GCaMP-ER2) were designed in the laboratory of Dr. Michael I. Kotlikoff (Cornell University) as part of Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).
The ~175 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-370F21 was obtained; containing the entire actin alpha 2 locus (Acta2) as well as ~92 kbp of 5' flanking sequences (including the complete Fas locus) and ~68 kbp of 3' flanking sequences (including the complete Stambpl1 locus). Using homologous recombination/BAC recombineering, a 2344 bp GCaMP-ER2-SV40pA construct (details below) was inserted into the ATG start site of the BAC Acta2 gene (replacing the initiation codon of Acta2 in exon 2). No other loci on the BAC were altered. The 11,612 bp BAC vector (pBACe3.6) contained a chloramphenicol selection cassette.
The resulting ~189 kbp modified BAC (Acta2BAC-GCaMP-ER2) was purified and then microinjected into the male pronucleus of C57BL/6J x SJL/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Acta2-GCaMP-ER2 founder line B25-6 was identified with high sensor expression in smooth muscle cells. The donating investigator reports that Acta2-GCaMP-ER2 transgenic mice from this founder line were backcrossed to C57BL/6J for a total of five generations, and then hemizygous males (black coat color) were sent to The Jackson Laboratory Repository in 2019. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin). The transgene insertion site(s) and transgene copy number were not characterized (July 2019).
GCaMP-ER2 is a genetically-encoded, endoplasmic reticulum (ER)-localized calcium indicator with low Ca2+ affinity and high dynamic range (Kd = 388μM, ΔFmax/F0 = 40) as described in Sun et al. 2019 Cell Death Dis. 10:396 [PMID:31113961] and summarized below.
GCaMP-ER2 is composed of (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability (RSET), a 13 residue peptide of chicken smooth muscle myosin light chain kinase (M13; target peptide for a Ca2+-bound CaM), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with specific mutations]), a rat calmodulin DNA fragment (CaM; aa 2-148 [with specific mutations]). First, the GCaMP3 variant G-GECO1.2 sequence was obtained and then modified to have low Ca2+ affinity and high dynamic range via introduction of two mutations in the CaM EF-hand loops (D24A, D129A) - resulting in the GCaMP variant GCaMP-L2. Targeting GCaMP-L2 to the ER was achieved by fusion of the N-terminal calreticulin ER targeting sequence (MLLSVPLLLGLLGLAVA) and the C-terminal ER retention signal (KDEL). Improved brightness in HeLa cells was shown via addition of a KL(AP)6 linker between CaM and KDEL - resulting in the final variant GCaMP-ER2.
|Allele Name||transgene insertion B25-6, Michael I Kotlikoff|
|Allele Type||Transgenic (Reporter)|
|Gene Symbol and Name||Tg(Acta2-GCaMP-ER2)B25-6Mik, transgene insertion B25-6, Michael I Kotlikoff|
|Strain of Origin||C57BL/6J x SJL/J|
|Molecular Note||Acta2-GCaMP-ER2 transgenic mice (also called Acta2-GCaMP-ER or Acta2 |
Mice hemizygous for the Acta2-GCaMP-ER2 transgene are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. When maintaining our live colony, hemizygous mice may be bred to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice (Stock No. 000664).
To date (July 2019), it has not been attempted to make this strain homozygous.
When using the Acta2-GCaMP-ER2 , Acta2BAC-GCaMP-ER2 , Acta2-GCaMP-ER mouse strain in a publication, please cite the originating article(s) and include JAX stock #032886 in your Materials and Methods section.
"MICE" means mouse strains, their progeny derived by inbreeding or crossbreeding, unmodified derivatives from mouse strains or their progeny supplied by The Jackson Laboratory ("JACKSON"). "PRODUCT(S)" means biological materials supplied by JACKSON, and their derivatives. "SERVICES" means projects conducted by JACKSON for other parties that may include but are not limited to the use of MICE or PRODUCTS. "RECIPIENT" means each recipient of MICE, PRODUCTS, or SERVICES provided by JACKSON including each institution, its employees and other researchers under its control. MICE or PRODUCTS shall not be: (i) used for any purpose other than internal research, (ii) sold or otherwise provided to any third party for any use, or (iii) provided to any agent or other third party to provide breeding or other services. Acceptance of MICE, PRODUCTS or SERVICES from JACKSON shall be deemed as agreement by RECIPIENT to these conditions, and departure from these conditions requires JACKSON’s prior written authorization.
MICE, PRODUCTS AND SERVICES ARE PROVIDED "AS IS". JACKSON EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS, IMPLIED, OR STATUTORY, WITH RESPECT TO MICE, PRODUCTS OR SERVICES, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR ANY WARRANTY OF NON-INFRINGEMENT OF ANY PATENT, TRADEMARK, OR OTHER INTELLECTUAL PROPERTY RIGHTS.
In case of dissatisfaction for a valid reason and claimed in writing by a purchaser within ninety (90) days of receipt of, PRODUCTS or SERVICES, JACKSON will, at its option, provide credit or replacement for the PRODUCT received or the SERVICES provided; JACKSON makes no other representations and this shall be the exclusive remedy of the purchaser. Please note specific policy for live mice.
Consistent with the requirement for a written understanding regarding animal care and use, the JACKSON Animal Care and Use Committee will review the animal care and use protocol(s) associated with any SERVICES to be performed at JACKSON, and JACKSON shall have ultimate responsibility and authority for the care of animals while on site or in JACKSON custody.
In no event shall JACKSON, its trustees, directors, officers, employees, and affiliates be liable for any causes of action or damages, including any direct, indirect, special, or consequential damages, arising out of the provision of MICE, PRODUCTS, or SERVICES, including economic damage or injury to property and lost profits, and including any damage arising from acts or negligence on the part of JACKSON, its agents or employees. Unless prohibited by law, in purchasing or receiving MICE, PRODUCTS, or SERVICES from JACKSON, purchaser or recipient, or any party claiming by or through them, expressly releases and discharges JACKSON from all such causes of action or damages, and further agrees to defend and indemnify JACKSON from any costs or damages arising out of any third party claims.
MICE, PRODUCTS or SERVICES are to be used in a safe manner and in accordance with all applicable governmental rules and regulations.
The foregoing represents the General Terms and Conditions applicable to JACKSON’s MICE, PRODUCTS or SERVICES. In addition, special terms and conditions of sale of certain MICE, PRODUCTS, or SERVICES may be set forth separately in JACKSON web pages, catalogs, price lists, contracts, and/or other documents, and these special terms and conditions shall also govern the sale of these MICE, PRODUCTS and SERVICES by JACKSON, and by its licensees and distributors.
Acceptance of delivery of MICE, PRODUCTS or SERVICES shall be deemed agreement to these terms and conditions. No purchase order or other document transmitted by purchaser or recipient that may modify the terms and conditions hereof, shall be in any way binding on JACKSON, and instead the terms and conditions set forth herein, including any special terms and conditions set forth separately, shall govern the sale of MICE, PRODUCTS or SERVICES by JACKSON.