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. 2022 Oct 17;31(6):1688–1704. doi: 10.1016/j.ymthe.2022.10.005

Figure 1.

Figure 1

Generation of transgenic mice expressing circular antisense against circSlc8a1 (cA-circSlc8a1) knocked down cardiac circSlc8a1 and developed phenotypic changes

(A) RNA sequencing heatmap of differentially expressed circular RNA profiles in the heart of 13 mice. The highest readcount was circSlc8a1 (mmu_circ_0000823), which was more than the sum obtained by adding the readcounts of the top 2–8 circRNAs. (B) Compared with the other organs analyzed, the heart expressed the highest level of circSlc8a1. n = 12; ∗∗p < 0.01 versus other organs. (C) The ratio of circSlc8a1 (circSlc) in the mouse heart compared with Slc8a1 (Slc) mRNA and ubiquitously expressed U6. n = 12; ∗∗p < 0.01 versus circSlc. (D) A diagram showing the sequence of the circular antisense against circSlc8a1 (cA-circSlc8a1) that was perfectly complementary to circSlc8a1. (E) Graphical representation of the three-dimensional structure of cA-circSlc8a1 and circSlc8a1, or cA-circSlc8a1 and Slc8a1 mRNA docked model visualized using PyMOL. (F) The back-splice junction of the ectopic mouse cA-circSlc8a1 was validated by Sanger sequencing. (G) Representative images of cA-circSlc8a1 expression with RNase R and RNase A treatment. A cA-circSlc8a1 construct targeting mouse circSlc8a1 was transfected into human HEK293T cells. The RNAs from vector or cA-circSlc8a1-transfected cells were treated with RNase R or RNase A followed by reverse transcription. The expression of cA-circSlc8a1 was validated by mouse-specific primers against cA-circSlc8a1. n = 6. (H) Representative images of cA-circSlc8a1 expression by using random or oligo(dT) primers for reverse transcription. A cA-circSlc8a1 construct targeting mouse circSlc8a1 was transfected into human HEK293T cells. The RNAs from vector or cA-circSlc8a1-transfected cells were reverse transcribed using random or oligo(dT) primers. The expression of cA-circSlc8a1 was validated by mouse-specific primers against cA-circSlc8a1. n = 6. (I) Representative images of unbound circSlc8a1 levels after transfection of cA-circSlc8a1 in HL-1 cells. The RNAs from vector or cA-circSlc8a1-transfected cells were reverse transcribed using specific reverse primers for circSlc8a1 followed by qPCR. n = 6. (J) The levels of circSlc8a1 and Slc8a1 mRNA in the heart of cA-circSlc8a1-transgenic mice. n = 8; ∗∗p < 0.01 versus negative. (K) Left, the body weight of cA-circSlc8a1(+) mice (cA-circSlc) was measured at different time points and compared with the age-matched cA-circSlc8a1(−) counterparts (negative) litters. ∗∗p < 0.01, n = 10. Right, a photo of cA-circSlc8a1(+) mice and their age-matched negative counterparts taken at the age of 10 months, showing an increase in the body weight in cA-circSlc8a1(+) mice.