BAC transgenic mice exhibit placental <i>CRH</i> expression and delayed parturition, which are eliminated by THE1B deletion.

(A) Schematic of human BAC clone RP11-366K18 (gray) with CRH shown to scale (red). (B) Expression of human CRH measured by qPCR in adult tissues and placenta. (n = 2–4 per group, P n = 28 +/+, n = 9 Tg[BAC1]/Tg[BAC1], P n = 17 Tg[BAC2]/Tg[BAC2], P = 0.0006 compared to +/+ by one-way ANOVA with post hoc Tukey’s multiple comparisons test. Error bars indicate standard deviation.) (D) Gestation length of litters hemizygous for Tg(BAC1) or wild-type control. Litters receiving Tg(BAC1) only from the father are born significantly later than wild-type control litters. (n = 20 +/+, n = 17 Tg[BAC1]/+, P = 0.0003 by unpaired two-tailed t-test. Error bars indicate the standard deviation.) (E) Schematic of 5-kb region containing CRH (gray) and THE1B (red). Blue arrows represent PAM sequences targeted by CRISPR/Cas9. (F) Sequenced deletions of the indicated transgenic founder animals compared to the Tg(BAC1) parent line. Red text, THE1B element. Blue text, PAM sequences indicated in (A). (G) Placental expression of human CRH at E18.5 measured by qPCR. (n = 3 Tg[BAC1]/+, n = 6 Tg[CR1]/+, P = 0.0006 by one-way ANOVA with post hoc Tukey’s multiple comparisons test. n = 5 Tg[CR2]/+, P = 0.0035 compared to Tg[BAC1]/+ by one-way ANOVA with post hoc Tukey’s multiple comparisons test. Error bars indicate the standard error of the mean.) (H) Hypothalamic expression of human CRH measured by qPCR. (n = 3 Tg[BAC1]/+, n = 4 Tg[CR1]/+, P = 0.0033 by one-way ANOVA with post hoc Tukey’s multiple comparisons test. n = 4 Tg[CR2]/+, P = 0.0013 compared to Tg[CR1]/+ by one-way ANOVA with post hoc Tukey’s multiple comparisons test. Error bars indicate the standard error of the mean.) (I) Gestation length of litters homozygous for Tg(CR1), homozygous for Tg(CR2), or wild-type control. The gestation length of THE1B-deleted transgenic litters is not significantly different from wild-type control litters. (n = 39 +/+, n = 15 Tg[CR1]/Tg[CR1], n = 7 Tg[CR2]/Tg[CR2], P = 0.0568 by one-way ANOVA. Error bars indicate standard deviation.) Numerical data for B, C, D, G, H, and I can be found in S1 Data. BAC, bacterial artificial chromosome; CRISPR/Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; E18.5, embryonic day 18.5; HT, hypothalamus; ND, not detected; ns, not significant; PAM, protospacer adjacent motif; qPCR, quantitative PCR; skelM, skeletal muscle; THE1B, transposon-like human element 1B.