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Fig. 1.—. Targeting vector construction and targeting strategy. (A) Mouse genome fragment (black box) from XhoI to BamHI, including Pou3f2 ΔGQP (red) in pBluescript SK(+) (left). Targeting vector was created using this fragment (right). Green box is Neor, orange box is DT-A, bold black line is mouse genome, and brown line is pBluescript SK(+). DT-A, diphtheria toxin fragment A; Neor, neomycin-resistance gene. (B) Mouse genome fragment from XhoI to BamHI including xPou3f2 (blue) in pBluescript SK(+) (left). This fragment was used for the targeting sequence (right). (C) Strategy for generating the xPou3f2 knock-in mouse. (D) Strategy for generating the Pou3f2 ΔGQP knock-in mouse. Yellow triangles indicate the sequence of loxP sites. Restriction sites are shown in the schematic sequence.
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Fig. 2.—. Weaning ratio. (A) Schematic of Pou3f2 genomic region in +/+ mice, two types of knock-in mice, and Xenopus tropicalis. Box shows Pou3f2 coding region, and straight lines indicate Pou3f2 upstream and downstream regions. Three homopolymeric amino acid repeats are shown in red, and DNA-binding domains are shown in gray. (B) Distribution of weaning ratio. Black and blue represent the weaning ratio of pups for +/+ and tro/tro dams, respectively. (C) Mean weaning ratio. ***P < 0.001 (Student’s t test). (D) Genotype ratio of pups from +/tro parents.
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Fig. 3.—. Retrieval test. (A) Example of the state in which females can retrieve and cannot retrieve. (B) Schematic of the test. (C) Distribution of the number of retrieved pups. Percentages and numbers of tested mice retrieving 3, 2, 1, or 0 pups are shown in each bar. (D) Latency to first contact in the retrieval test. Values represent mean ± SEM. (E) Box plot of retrieval latency for each pup. Top and bottom edge of box show the first and third quantile, respectively. Line in the box indicates median value. Top and bottom edge of line indicate maximum and minimum, respectively. n = 19 for +/+ and n = 16 for tro/tro.
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Fig. 4.—. Habituation–dishabituation test. (A) Schematic of habituation–dishabituation test. (B) Paradigm and sequence for presenting the young mouse A or B. Each interval is 1 min. (C) Total sniffing time at each section. Values represent mean ± SEM. **P < 0.01, ***P < 0.001 (Student’s t-test).
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Fig. 5.—. Quantitative immunohistochemistry for TH. (A) Fluorescence intensities were measured in transverse sections located approximately 0.74 mm rostral from bregma, including DA-rich regions, the neostriatum, and nucleus accumbens. Abbreviations (Franklin and Paxinos 2008): STlat, lateral part of the neostriatum; STmed, medial part of the neostriatum; AcbS, shell of the nucleus accumbens; AcbC, core of the nucleus accumbens; Tu, olfactory tubercle; M1, primary motor cortex; S1, primary sensory cortex; DI, dysgranular insular cortex; Pir, piriform cortex; and sepN, septal area. (B–D) Quantitative distribution, comparative analysis, and enlarged ventral striatum region for +/+ and tro/tro, respectively. (E–G) Quantitative distribution, comparative analysis, and enlarged ventral striatum region for +/+ and Δ/Δ, respectively. Values shown on graphs represent the mean ± SEM. *P < 0.05; **P < 0.01.
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Fig. 6.—. Quantitative immunohistochemistry for TPH2. (A) Fluorescence intensities were measured in transverse sections located approximately 4.60 mm caudal from bregma, including 5-HT-rich region, the raphe nucleus. Abbreviations (Franklin and Paxinos 2008): DRD, dorsal part of the dorsal raphe nucleus; DRV, ventral part of the dorsal raphe nucleus; DRL, lateral part of the dorsal raphe nucleus; MnR, median raphe nucleus; PMnR, paramedian raphe nucleus; ECIC, external cortex of the inferior colliculus; mRt, mesencephalic reticular formation; LL, lateral lemniscus; xscp, decussation of the superior cerebellar peduncle; RtTg, reticulotegmental nucleus of the pons. (B–D) Quantitative distribution, comparative analysis, and enlarged the raphe nucleus for +/+ and tro/tro, respectively. (E–G) Quantitative distribution, comparative analysis, and enlarged the raphe nucleus for +/+ and Δ/Δ, respectively. Values shown on graphs represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
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Fig. 7.—. Reduction rates of TH and TPH2. (A) Reduction rate of TH in five regions (STlat, STmed, AcbS, AcbC, and Tu). “cont.ave” shows average of cortex regions (M1, S1, DI, and Pir). (B) Reduction rate of TPH2 in five regions (DRD, DRV, DRL, MnR, and PMnR). “cont.ave” shows average of other regions (ECIC, mRt, LL, xscp, and RtTg). Reduction rates were calculated as percentage of tro/tro (blue bars) or Δ/Δ (red bars) against +/+.
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