B6.Cg-Adcyap1^{tm1.1(cre)Hze}/ZakJ

Stock number: 030155
Product name: Adcyap1-2A-Cre knock-in (PACAP-2A-Cre , Adcyap1-2A-Cre-D , PACAP-2A-Cre-D)
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

Adcyap1-2A-Cre knock-in mice are designed to have Cre recombinase expression directed to adenylate cyclase activating polypeptide 1-expressing cells. Cre expression and activity in adult mice recapitulates that of the endogenous Adcyap1 gene in the brain. These mice may be used to generate conditional mutations for studying this neuropeptide/neurotrophin in aspects of neuronal function, including neurotransmission, neuromodulation and neuroendocrine stress responses and heat responses.

Detailed description

The adenylate cyclase activating polypeptide 1 protein (ADCYAP1 or PACAP) encoded by the Adcyap1 gene is a neuropeptide/neurotrophin that functions as a hypophysiotropic hormone, neurotransmitter and neuromodulator, and also plays a role in paracrine and autocrine regulation of certain cell types. In addition, warm-sensitive neurons within the preoptic hypothalamus that coordinate the diverse behavioral and autonomic responses to heat are molecularly defined by co-expression of the neuropeptides BDNF and ADCYAP1.

The Adcyap1-2A-Cre knock-in mutation has a F2A sequence and Cre recombinase gene inserted inserted immediately upstream of the Adcyap1 translational STOP codon - this is designed to have the endogenous promoter/enhancer elements directing cre expression to ADCYAP1+ cells. When Adcyap1-2A-Cre mice are bred with mice containing lox-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Adcyap1-expressing cells in the offspring.

The donating investigators report Cre recombinase expression and activity recapitulates the endogenous Adcyap1 gene in adult mice. Expression is observed in preoptic hypothalamus of adult mice. Additionally, expression is enriched in restricted populations within the olfactory areas, hippocampus, striatum, thalamus, midbrain, pons and medulla, and scattered within the isocortex and hypothalamus.
Importantly, the donating investigators report that Adcyap expression during development results in widespread recombination of floxed conditional alleles integrated into the genome (e.g., Gt(ROSA)26Sor reporters) - and they suggest genetic access to ADCYAP1+ cells in adult brain may benefit by using Cre-dependent AAV injections. The donating investigators did not examine cre expression in tissues other than brain. This knock-in allele is designed to avoid disrupting expression of the endogenous protein; although Adcyap1 expression from the Adcyap1-2A-Cre allele has not been evaluated to date (February 2017). Homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.

For original characterization information, see images at the Allen Institute for Brain Science website (Adcyap1-2A-Cre images).

Development

The bicistronic Adcyap1-2A-Cre knock-in allele (also called PACAP-2A-Cre, Adcyap1-2A-Cre-D, PACAP-2A-Cre-D) was created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a F2A sequence and a Cre recombinase gene inserted immediately upstream of the translational STOP codon of the adenylate cyclase activating polypeptide 1 gene (Adcyap1) on chromosome 17. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Adcyap1 sequence spanning intron 1 through part of exon 5 (with a frt3 site in intron 4) up to but not including the endogenous stop codon, a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), a Cre recombinase gene (cre), a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene (Hygro2) with SV40 polyA signal, and an AttP site. This construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked sequences (PGK-Neo-pA::frt5::RNA splice acceptor::3'hygro-pA) and replace it with the recombined AttB/AttP site (AttL).

The resulting Adcyap1-2A-Cre mice were backcrossed to C57BL/6 wildtype mice for at least seven generations (and the PhiC31 transgene was removed), and then sent to Dr. Zachary A. Knight (University of California, San Francisco) in 2014. Thereafter, Dr. Knight backcrossed the colony to C57BL/6J wildtype mice for at least five additional generations prior to sending male mice with black coat color to The Jackson Laboratory Repository in 2017. Upon arrival, males are used to cryopreserve sperm. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6J background during backcrossing.

Allele

Symbol: Adcyap1^{tm1.1(cre)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5635438
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Adcyap1
Marker name: adenylate cyclase activating polypeptide 1
Chromosome: 17
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre expression is observed in preoptic hypothalamus of adult mice. Additionally, expression is enriched in restricted populations within the olfactory areas, hippocampus, striatum, thalamus, midbrain, pons and medulla, and scattered within the isocortex and hypothalamus.
Molecular note: The targeting vector contains from 5' to 3', a partial Adcyap1 sequence spanning intron 1 through part of exon 5 (with an FRT3 site in intron 4) up to but not including the endogenous stop codon, a viral 2A oligopeptide sequence (F2A; mediates ribosomal skipping), a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK-Neo, an FRT5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene with SV40 polyA signal, and an AttP site. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL).