B6.FVB-Bdnf^em1(cre)Zak/J

Stock number: 030189
Product name: BDNF-2A-Cre knock-in
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

BDNF-2A-Cre knock-in mice are designed to have Cre recombinase expression directed to brain derived neurotrophic factor-expressing cells. Cre activity recapitulates that of the endogenous Bdnf gene in the brain. These mice may be used to generate conditional mutations for studying this neuropeptide/neurotrophin in aspects of neuronal function, including growth/differentiation of neurons and synapses, neurogenesis, synaptic transmission, learning, long-term memory, heat responses and human disease.

Detailed description

The brain-derived neurotrophic factor protein (BDNF) encoded by the Bdnf gene is a neuropeptide/neurotrophin with pivotal roles in neuronal functions, including growth/differentiation of neurons and synapses, neurogenesis, synaptic transmission, learning and long-term memory. Abnormal BDNF expression/function may also be linked to human diseases such as depression, schizophrenia, obsessive-compulsive disorder, Alzheimer's disease, Huntington's disease, Rett syndrome and dementia. 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 BDNF-2A-Cre knock-in mutation has a T2A sequence and Cre recombinase gene inserted immediately upstream of the Bdnf translational STOP codon - this is designed to have the endogenous promoter/enhancer elements direct cre expression to BDNF+ cells. When BDNF-2A-Cre mice are bred with mice containing lox-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Bdnf-expressing cells in the offspring.

The donating investigator reports Cre recombinase activity largely recapitulates the endogenous Bdnf gene. Specifically, they report recombinase activity mirrors endogenous BDNF in brain regions such as ventromedial, paraventricular and preoptic hypothalamus, as well as cortex, and CA1 and dentate gyrus of the hippocampus. The donating investigator 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 Bdnf expression from the BDNF-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.

Development

The bicistronic BDNF-2A-Cre knock-in allele was created in the laboratory of Dr. Zachary A. Knight (University of California, San Francisco) using homologous recombination (aided by targeted CRISPR/cas9 endonuclease activity) to insert a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping) and a Cre recombinase gene (cre) just upstream of the endogenous STOP codon of the brain derived neurotrophic factor locus (Bdnf) on chromosome 2. The targeting vector, Cas9 endonuclease and single guide RNA were mixed and injected into the pronuclei of FVB/NJ fertilized zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups (identified by PCR and sequencing) were bred to C57BL/6J inbred mice for germline transmission. The BDNF-2A-Cre colony (containing no off-target insertions of the vector) was backcrossed onto C57BL/6J for a total of at least nine generations, and then males with black coat color (see note below) were sent to The Jackson Laboratory Repository in 2017. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Regarding coat color, the donating investigator reported that despite nine generations of backcrossing with C57BL/6J, the BDNF-2A-Cre mice were almost always brown/agouti, with rare incidence of black coat color. In 2017, they chose mice with black coat color to send to The Jackson Laboratory Repository.

Allele

Symbol: Bdnf^em1(cre)Zak
Name: endonuclease-mediated mutation 1, Zachary Knight
MGI accession ID: MGI:5824836
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Marker symbol: Bdnf
Marker name: brain derived neurotrophic factor
Chromosome: 2
Strain of origin: FVB/NJ
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase activity largely recapitulates the endogenous Bdnf gene in brain regions such as ventromedial, paraventricular and preoptic hypothalamus, as well as cortex, and CA1 and dentate gyrus of the hippocampus.
Molecular note: The bicistronic BDNF-2A-Cre knock-in allele was created using homologous recombination (aided by targeted CRISPR/cas9 endonuclease activity) to insert a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping) and a Cre recombinase gene (cre) just upstream of the endogenous STOP codon of the brain derived neurotrophic factor locus (Bdnf). The targeting vector, Cas9 endonuclease and single guide RNA were mixed and injected into the pronuclei of FVB/NJ fertilized zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6J inbred mice for germline transmission.