C57BL/6-Tg(CAG-RFP/EGFP/Map1lc3b)1Hill/J

Stock number: 027139
Product name: CAG-RFP-EGFP-LC3
Research areas: Apoptosis Research, Research Tools

Description

CAG-RFP-EGFP-LC3 transgenic mice express RFP and EGFP in a pH-dependent manner in phagocytic cells following ischemic injury. They are useful for studying the initiation, progression, and resolution of autophagy in a variety of tissues following ischemic injury.

Detailed description

CAG-RFP-EGFP-LC3 transgenic mice have CAG promoter/enhancer sequences driving expression of a red fluorescent protein (RFP), an enhanced green fluorescent protein (EGFP), and a microtubule-associated protein 1 light chain 3 alpha (Map1lc3a or LC3) gene. LC3 serves as a marker for phagosomes in a wide variety of tissues. The dual expression of two fluorophores allows tracking of different types of phagocytic cellular compartments depending on the acidity of the environment. RFP is stable in acidic pH (pK_a 4.5) while EGFP (pK_a 5.9) is quenched in the acidic lysosomal environment, allowing autophagosomes to be distinguished from autolysosomes. Combined GFP and RFP fluorescence yields a yellow signal within high pH phagophores and autophagosomes, while EGFP is quenched in autolysosomes, and they emit only an RFP signal. Following ischemia-reperfusion injury, EGFP expression reaches a peak at 24 hours and returns to baseline levels at 3 days. Meanwhile, RFP expression also peaks at 24 hours but persists at high levels for 3 days before returning to baseline levels by 7 days. CAG-RFP-EGFP-LC3 mice are useful for studying the progression and recovery from ischemic injury in a variety of tissues. Hemizygotes are viable and fertile. The donating investigator did not try to make a homozygous colony.

Development

The CAG-RFP-EGFP-LC3 transgene was designed with a human cytomegalovirus immediate early promoter enhancer with chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from the pCAGGS vector) driving expression of a red fluorescent protein (RFP), an enhanced green fluorescent protein (EGFP), and a microtubule-associated protein 1 light chain 3 alpha (Map1lc3a or LC3) gene. The transgene was microinjected into fertilized C57BL/6 oocytes and the donating investigator reported that founder mice were bred to C57BL/6 mice (see SNP note below). Upon arrival at The Jackson Laboratory, transgenic mice were bred to C57BL/6J inbred mice (Stock No. 000664) to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. One of the 27 markers throughout the genome was segregating from an unknown source. All 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

 

Allele

Symbol: Tg(CAG-RFP/EGFP/Map1lc3b)1Hill
Name: transgene insertion 1, Joseph Hill
MGI accession ID: MGI:5635828
Generation method: Transgenic
Attributes: Reporter; Inserted expressed sequence
Marker symbol: Tg(CAG-RFP/EGFP/Map1lc3b)1Hill
Marker name: transgene insertion 1, Joseph Hill
Chromosome: UN
Strain of origin: C57BL/6
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,; Map1lc3b,microtubule-associated protein 1 light chain 3 beta,mouse, laboratory
Site of expression: Expression occurs in a variety of tissues.
Molecular note: The transgene was designed with a human cytomegalovirus immediate early promoter enhancer with chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from the pCAGGS vector) driving expression of a fusion of a red fluorescent protein (RFP) with a Strep-tag sequence to facilitate isolation, an enhanced green fluorescent protein (EGFP), and a microtubule-associated protein 1 light chain 3 beta (Map1lc3b or LC3) gene.