B6N.Cg-Tg(Arc-tTA,-EGFP/Capzb*)10Mkyph/J

Stock number: 035474
Product name: AiCE-Tg
Research areas: Neurobiology Research, Research Tools

Description

AiCE-Tg transgenic mice express an EGFP-tagged Capzb cDNA (β2 splice variant) in dendritic spines and also expresses doxycycline-conditional expression of the tetracycline-controlled transactivator (tTA) protein - all under the control of the mouse Arc (activity regulated cytoskeletal-associated protein) promoter. EGFP fluorescence intensity reflects levels of visual or somatosensory stimulation to dendritic spines and cellular activity. This strain allows direct visualization of Capzb (β2 splice variant) expression in the brain.

Detailed description

AiCE-Tg transgenic mice have a 7kb promoter sequence from mouse Arc (activity regulated cytoskeletal-associated protein) driving expression of a fused EGFP-Capzb and a tetracycline-controlled transactivator (tTA) protein. Arc is known to be expressed in activated neurons. CapZ (F-actin capping protein) was focused on as a decreased spot in the 2D-DIGE analysis of the hippocampi of a rodent model of dementia [PMID:20545768]. CapZ protein levels are considered to be regulated by degradation [PMID:20884878] and increased in LTP-induced DG layers. It is also reported to be reduced in human brains with pathological conditions that include memory impairment (Fetal Down's-S, AD). EGFP-CapZ accumulates in a part of dendritic spines, which is suggested to be "active spines" in active neurons [PMID:35297954, PMID:32943708].

AiCE-Tg hemizygous mice have levels of EGFP/CapZ fusion protein that are ~10% of the endogenous CapZ protein levels. EGFP fluorescence in the footpads and adult brains is high/robust - bright enough to be detected by the naked eye through adequate filters under blue light. The footpad expression is believed to be from their peripheral neurons (characterized by distinctive shapes such as Pacinian corpuscles).

AiCE-Tg hemizygotes are viable and fertile with no obvious phenotype including the score of contextual fear conditioning test. To date (Jan 2023), the donating investigator has generated at least one probable AiCE-Tg homozygous male that seemed unhealthy and had a low rate of successful mating.

When mated to a strain carrying a gene of interest under the regulatory control of a tetracycline-responsive promoter element (TRE or tetO), the donating investigator confirmed expression of the target gene resembles that of Arc. EGFP-CapZ-expressing neurons overlapped, but not identically, to those of Arc, suggesting that they are differentially regulated in their degradation (PMID:20884878). The expression was limited to neurons.
Furthermore, the donating investigator reports EGFP expression is very bright and detectable by direct fluorescence. In experiments using immunohistochemical staining, neurons expressing EGFP generally corresponded to ARC-positive cells, although they do not match in intensity. In groups of cells with high expression intensity for each marker, the expression of the other marker is often not detectable.

The donating investigator requests that when using this mouse line in a publication or an official presentation, please refer to the mice as AiCE-Tg and include the JAX Stock No. 035474 in your Materials and Methods section.

Development

The transgenic construct contains the 7kb mouse Arc (activity regulated cytoskeletal-associated protein) promoter, the tetracycline-controlled transactivator (tTA), a P2A self-cleaving peptide, a cDNA encoding an EGFP/modified-Capzb (capping actin protein of muscle Z-line subunit beta; β2 splice variant, MGI:104652) fusion protein and a polyA sequence. The EGFP-CapZ retains CapZ's native C-terminus region that caps F-actin and binds other major binding proteins. The β2 splice variant of CapZβ is expressed primarily in the brain and less so in striated muscle, which has CapZ in the Z disc. The donating investigator (Dr. Yamada) reports the linearized construct was introduced by pronuclear injection into fertilized eggs from "DBF1xB6N" mice (a hybrid created from crossing B6D2F1/Crl [(C57BL/6NCrl x DBA/2NCrl)F1] with C57BL/6NCrl). AiCE-Tg founder 10 (formerly 53) was identified to have 100 copies of the transgene (chromosomal insert location is not known) and was mated to C57BL/6. The strain was then maintained on a C57BL/6 genetic background for more than 14 years (backcrossed 4 generations to C57BL/6NCrl, then backcrossed 3 generations to C57BL/6JJmsSlc, then backcrossed 10 generations to C57BL/6NCrl and then transgenic mice were bred to wildtype (non-transgenic) siblings each generation for 10 generations), during which time no obvious changes/loss in fluorescence intensity were noted. In 2024, the donating investigator sent 2 hemizygous males (black coat color) to The Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

Allele

Symbol: Tg(Arc-tTA,-EGFP/Capzb*)10Mkyph
Name: transgene insertion 10, Maki Yamada
MGI accession ID: MGI:7344056
Generation method: Transgenic
Attributes: Reporter; Inserted expressed sequence; Transactivator
Synonyms: AiCE-Tg; C57BL/6N-Tg(Arc-tTA/egfp/capzb*2)100Mkyph
Marker symbol: Tg(Arc-tTA,-EGFP/Capzb*)10Mkyph
Marker name: transgene insertion 10, Maki Yamada
Chromosome: UN
Strain of origin: C57BL/6N
Molecular note: The transgenic construct contains sequence coding for a tetracycline-controlled transactivator (tTA), followed by a P2A self cleaving peptide, sequence coding for an EGFP/modified Capzb (capping actin protein of muscle Z-line subunit beta) fusion protein, and polyA signal sequence under the control of the 7 kb mouse Arc (activity regulated cytoskeletal-associated protein) promoter. EGFP-CapZ retains native C-terminus region that caps F-actin and binds other major binding proteins. The beta2 splice variant is expressed primarily in the brain and less so in striated muscle, which has Capz in the Z disc.