Influence of poly(A) sequences and genetic elements on iCre expression and induction of a hibernation-like state in mice

Activation of hypothalamic QRFP-producing neurons (Q neurons) induces a hibernation-like state in mice, termed Q neurons–Induced Hypometabolism (QIH). To achieve reliable chemogenetic induction of QIH, precise expression of iCre in Q neurons is essential. Polyadenylation [poly(A)] cassettes, commonly included in knock-in constructs, not only stabilize transcripts but also influence transcription termination and expression specificity. Here we systematically compared three modifications of the Qrfp-iCre allele: (1) replacing the SV40 poly(A) with a bovine growth hormone (bGH) poly(A), (2) introducing bicistronic expression systems (T2A or IRES), and (3) deleting the poly(A) cassette to force reliance on the endogenous Qrfp poly(A) signal. The bGH poly(A) disrupted cell-type specificity and attenuated QIH, bicistronic constructs yielded largely specific but weak expression, and poly(A) deletion partially restored specificity and, in homozygotes, QIH depth, but shortened the duration. These findings demonstrate that poly(A) cassette choice is not a neutral design element but a critical determinant of both molecular and physiological outcomes, providing practical guidance for the design of neuronal knock-in models.