C57BL/6J-Htr2a^em3(cre)Blr/J

Stock number: 038936
Product name: Htr2a-A242S-EGFP-CT-IRES-Cre , Htr2a^{A242S-EGFP-Cre}
Research areas: Neurobiology Research, Research Tools

Description

Htr2a^{A242S-EGFP-Cre} (Htr2a-A242S-EGFP-CT-IRES-Cre) mice express a humanized version of the mouse Htr2a gene in which a single point mutation in alanine residue 242 was replaced by a serine from the human HTR2A gene. An EGFP fluorescent reporter was also inserted into the C-terminus (CT) followed by IRES-Cre. The line enables the independent identification of both HTR2A-EGFP receptors and HTR2A-containing cells and represents a useful tool for elucidating the molecular, cellular, pharmacological, physiological, behavioral, and other actions of psychedelic drugs in vivo.

Detailed description

HTR2A (5-hydroxytryptamine 2A receptor) plays a role in the pathogenesis of several psychiatric disorders, including schizophrenia, depression, Parkinson's Disease psychosis, and anxiety disorders. This receptor is also the primary molecular target for psychedelic drugs and non-psychedelic therapeutics.

Htr2a^{A242S-EGFP-Cre} (Htr2a-A242S-EGFP-CT-IRES-Cre) mice express a humanized version of the mouse Htr2a gene in which a single point mutation in alanine residue 242 was replaced by a serine from the human HTR2A gene. An EGFP fluorescent reporter was also inserted into the C-terminus (CT) followed by IRES-Cre. The point mutation enhances the affinity and potency of a variety of psychedelic and non-psychedleic drugs to levels similar to those seen at the human receptor.

The Roth lab (University of North Carolina at Chapel Hill) produced a series of three strains that express Cre recombinase from the mouse Htr2a promoter: Htr2a^EGFP-CreERT2, expressing mouse HTR2A protein, EGFP, and tamoxifen-inducible Cre (Stock No. 038934); Htr2a^Cre, constitutively expressing mouse HTR2A protein and Cre (Stock No. 038935), and Htr2a^{A242S-EGFP-Cre}, constitutively expressing humanized HTR2A and Cre (Stock No. 038936). HTR2A protein is expressed from the targeted gene in a pattern similar to that of wildtype animals in all three of these knock-in strains, as determined by immunohistochemistry and Western blot.

Fluorescent reporter expression patterns were found to differ among the three lines. Results also varied depending on whether a viral vector or crosses with Ai9 mice (Stock No. 007909) were used in recombinant tdTomato reporter expression tests. These differences were attributed differences in timing and efficiencies of the experimental components. Full details may be found in the primary reference (Chiu, 2023). The authors note that it will be important to validate whether reporter expression patterns correspond with HTR2A receptor distributions in the same brain region.

Development

A cDNA encoding EGFP (flanked on both ends by a serine- and glycine- rich linker) was inserted into the C-terminus (CT) of the mouse Htr2a gene after residue 452 (encoded by exon 3; chr14:74706337 in the GRCm38/mm10 genome assembly) and an IRES-Cre cassette was placed between the stop codon and the 3'UTR (chr14:74706397). A single humanizing A242S (nucleotide: GCA to TCC) mutation was also introduced to exon 3 of the mouse Htr2a gene. The allele was created using CRISPR/Cas9 methodologies in C57BL/6J embryos. Resultant mice were backcrossed to C57BL/6J for 10 generations by the donating laboratory to help remove any off-target mutations.

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve C57BL/6J (Stock No. 000664).

Allele

Symbol: Htr2a^em3(cre)Blr
Name: endonuclease-mediated mutation 3, Bryan L Roth
MGI accession ID: MGI:7543067
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Reporter; Humanized sequence
Synonyms: Htr2a^{A242S-EGFP-Cre}; Htr2a^{em3(EGFP, cre)Blr}; Htr2a-A242S-EGFP-CT-IRES-Cre
Marker symbol: Htr2a
Marker name: 5-hydroxytryptamine (serotonin) receptor 2A
Marker synonyms: 5Ht-2; 5-HT2A; 5-HT2A receptor; HTR2; Htr-2
Chromosome: 14
Strain of origin: C57BL/6J
Molecular note: A cDNA encoding EGFP (flanked on both ends by a serine- and glycine- rich linker) was inserted into the C-terminus (CT) of the mouse Htr2a gene after residue 452 (encoded by exon 3; chr14:74706337 in the GRCm38/mm10 genome assembly) and an IRES-Cre cassette was placed between the stop codon and the 3' UTR (chr14:74706397). A single humanizing A242S (nucleotide: GCA to TCC) mutation was also introduced to exon 3 of the mouse Htr2a gene. The allele was created using CRISPR/cas9 methodologies in C57BL/6J embryos.