Functional Interrogation of a Depression-Related Serotonergic Single Nucleotide Polymorphism, rs6295, Using a Humanized Mouse Model

The serotonin 1A receptor (5-HT1A) system has been extensively implicated in modulating mood and behavior. Notably, 5-HT1A levels in humans display remarkable variation, and differences in receptor levels have been linked with a variety of psychiatric disorders. Further, reduction of receptor levels by 30-50% in mice suggests that changes in receptor levels that model existing human variation are sufficient to drive behavioral alterations. As a result, genetic mechanisms that modulate human 5-HT1A levels may be important for explaining individual differences in mood and behavior, representing a potential source of psychiatric disease risk. One common genetic variant implicated in differential 5-HT1A levels is the G/C single nucleotide polymorphism (SNP) rs6295, located upstream of the human 5-HT1A gene. This SNP differentially binds the transcription factor, NUDR/Deafl, leading to cell-type specific effects on transcription in vitro. To investigate the direct effects of this SNP in the heterogeneous cellular context of the brain, we generated humanized transgenic mice using a design that maximized the local transcriptional landscape of the human HTR1A gene while also controlling for effects of genomic insertion location. We integrated a 180 kb human bacteria artificial chromosome (BAC) transgene containing G- and C-alleles of rs6295 flanked by FRT or loxP sites. Subsequent deletion of each allele by Cre- or Flp-recombinase resulted in rs6295G and C alleles in the same genomic location. These alleles were bred onto a 5-HT1A null mouse such that the human BAC was the sole source of 5-HT1A in these mice. We generated three separate lines, two of which had detectable human 5-HT1A levels in the brain, although none displayed expression in the raphe. Of these, one line exhibited rs6295-dependent differences in 5-HT1A levels and differences in behavior, even though the overall levels were considerably lower than native expression levels. The line-dependent effect of rs6295 on protein levels and behavior may depend upon differences in background genetic factors or different insertion sites across each line. This work confirms that relatively subtle differences in 5-HT1A levels can contribute to differences in behavior and highlights the challenges of modeling human noncoding genetic variation in mice.