STOCK Htr3a^{tm1.1(flpo)Rudy}/J

Stock number: 030755
Product name: Htr3a-ires-Flpo knock-in , Htr3a-flpo , 5HT3aR-Flpo
Research areas: Neurobiology Research, Research Tools

Description

Htr3a-ires-Flpo knock-in mice (also called Htr3a-flpo or 5HT3aR-Flpo) are designed to have the endogenous serotonin receptor 3A (Htr3a; 5HT3a, 5HT3aR) promoter/enhancer directing expression of an optimized FLP recombinase, as well as to retain endogenous Htr3a expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function, fate mapping and/or circuit dissection of 5HT3aR+ neurons in the cerebral cortex (e.g., 5HT3aR+ cortical GABAergic interneurons).

Detailed description

Serotonin receptor 3A (Htr3a; 5HT3a, 5HT3aR) is expressed in a subpopulation of GABAergic interneurons in the cerebral cortex.

The Htr3a-ires-Flpo knock-in allele (also called Htr3a-flpo or 5HT3aR-Flpo) has an IRES sequence and nuclear-localized FLPo recombinase gene inserted after the STOP codon of the Htr3a locus. This is designed to have the endogenous Htr3a promoter/enhancer driving FLPo expression, as well as to retain endogenous Htr3a expression. Specific details are below.

Mice heterozygous or homozygous for this Htr3a-ires-Flpo knock-in allele are viable and fertile with normal breeding, and no reported gross phenotypic or behavioral abnormalities. While Htr3a mRNA expression pattern/levels (in situ hybridization) in somatosensory cortex from Htr3a-ires-Flpo heterozygotes showed no differences compared to wildtype, the donating investigator reports homozygous tissues were not tested to date (January 2019).

When Htr3a-ires-Flpo are bred with mice containing frt-flanked sequences, FLPo-mediated recombination results in deletion of frt-flanked sequences in 5HT3aR expressing cells of the offspring.
For example, when bred to Rosa26^FSF-tdTomato (Ai65F; see Stock No. 032864), the resulting Htr3a-flpo;Ai65F mice are useful for selective targeting of 5HT3aR+ cortical GABAergic interneurons (i.e., primarily VIP or Reelin expressing interneurons that arise from the caudal ganglionic eminence [CGE]). In addition, the donating investigator reports the extent of fluorescent reporter labeling can be enhanced by having two copies of the FLPo driver (e.g., Htr3a-flpo;Ai65F mice that are homozygous for Htr3a-ires-Flpo). [Schuman et al. 2019 J Neurosci 39:125]

Development

The Htr3a-ires-Flpo knock-in allele (also called Htr3a-flpo or 5HT3aR-Flpo) was created in the laboratory of Dr. Bernardo Rudy (New York University) to have an IRES sequence and a FLPo recombinase gene inserted into the 3'UTR immediately following the STOP codon of the 5-hydroxytryptamine (serotonin) receptor 3A gene (Htr3a) on chromosome 9. The specific details are below.

The targeting vector contained, from 5' to 3', an internal ribosome entry site sequence (IRES; allows translation initiation in the middle of an mRNA sequence), the FLPo recombinase sequence (with nuclear localization signal at its 5' end), a bovine growth hormone polyA sequence and a lox2272-flanked PGK-Neo-bGHpA cassette. The construct was electroporated into B6.Cg-Thy1^a-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to C57BL/6J-congenic CMV-Cre mice (Stock No. 006054) for germline removal of the PGK-Neo-bGHpA cassette. The resulting Htr3a-ires-Flpo mice were bred with C57BL/6J wildtype mice for at least one generation (and the CMV-Cre transgene was removed) by the donating lab (see SNP note below). Htr3a-ires-Flpo mice were subsequently bred together for several generations, and then homozygous males (black coat color) were sent to The Jackson Laboratory Repository in 2019. Upon arrival, males are used to cryopreserve sperm. To establish our live Htr3a-ires-Flpo 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 Htr3a-ires-Flpo female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin). Additionally, the mice sent to The Jackson Laboratory Repository genotyped as homozygous for the Thy1^b allele (Thy1.2) that is normally present for C57BL/6 inbred mice.

In 2019 a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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. Four of the 43 markers throughout the genome (on Chromosomes 5, 7, 8, and 11) were segregating with an unknown source.

Allele

Symbol: Htr3a^{tm1.1(flpo)Rudy}
Name: targeted mutation 1.1, Bernardo Rudy
MGI accession ID: MGI:6284821
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Htr3a-ires-Flpo
Marker symbol: Htr3a
Marker name: 5-hydroxytryptamine (serotonin) receptor 3A
Marker synonyms: 5HT3; 5-HT3; 5-HT-3; 5-HT3 receptor; 5-HT3A; 5HT3R; 5-HT3R; HTR3
Chromosome: 9
Strain of origin: B6.Cg-Thy1^a
Molecular note: The targeting vector contains, from 5' to 3', an internal ribosome entry site sequence (IRES), the FLPo recombinase sequence (with nuclear localization signal at its 5' end), a bovine growth hormone polyA sequence and a lox2272-flanked PGK-Neo-bGHpA cassette inserted into the 3'UTR immediately following the STOP codon of the gene. Cre-mediated recombination removed the floxed neo cassette.