Introduction The X-inactivation center (Xic) controls X-chromosome inactivation (XCI) and is enriched for genes that produce long noncoding RNAs (ncRNA) (Figure 1A) [1], [2], [3]. Several have been shown to regulate XCI. The 17-kb Xist RNA is induced at the onset of XCI and silences the X-chromosome as the RNA spreads in cis along the chromosome. Xist expression is controlled both negatively and positively [4], [5], [6], [7], . Xist's antisense partner, Tsix, controls X-chromosome counting and allelic choice, blocking Xist induction on the future active X [8], [9], [11]. The upstream locus, Xite, is required to sustain Tsix expression on the future active X during XCI [12], [13]. Two other noncoding RNAs, Jpx and RepA, function as activators and are required for transcriptional induction of Xist RNA [7], [14]. Together, these five noncoding genes span 95% as judged by phase optics (for qRT-PCR in Figure 2C); RNA for qRT-PCR of Tsx in Figure 2B was also used in Reference 24. RNA from the fractionated cell populations was isolated using Trizol (Invitrogen). RT-PCR and real-time PCR analysis Tissues from 2–4 different wildtype male and female C57BL/6J animals (2 months old) were immediately placed in Trizol after dissection, then homogenized using a Qiagen TissueLyser, and the RNA was cleaned up using the RNeasy Mini kit (Qiagen). All RNA was treated with Turbo DNAse (Ambion) following the ‘rigorous’ protocol. Reverse transcription of RNA to cDNA was performed using SuperScript III (Invitrogen), 1 µg RNA and random hexamers. All cDNA was diluted 1∶5 prior to qPCR, except for cDNA from male and female animal tissues, which was not diluted. For real-time PCR analyses, Tsx cDNA (exons 1–7) was amplified using two primer sets: Tsx B10 and Tsx B11 [17] and Tsx 1 5′-ATTAAGCAGGCAGGCAGAAA-3′; Tsx 2 5′-TGCGGTGATTTTCATTTTGA-3′. For transcript amplification in PS (Figure 2D) we used a reverse primer (Tsx 3.5L; within exon 4) and 4 different forward primers (RPS-F, RPL-F, RPLb-F, RPLa-F; upstream of exon 1). ß-actin primers: 5′- CCGTGAAAAGATGACCCAG-3′ (F) and 5′-TAGCCACGCTCGGTCAGG-3′ (R). Tsx primers: 3.5L 5′-AGCTTGGCAAGTGTCCTC-3′; RPS-F 5′-TACCCTAGCTGAAGGAAAAT-3′; RPL-F 5′- ATGGTTGGAAGATCTAATACCT-3′; RPLa-F 5′-CAACCACTGTCCCCTTCCTA-3′; and RPLb-F 5′-CACCCCAGCAGAGAGAAAAG-3′. LINE-1 primers: 5′-GTCTGGTGTTTGGACCTCCT-3′ (F); 5′-CCGACATGTACGACTCCAGA-3′ (R). Standard PCR reactions for Tsx were cycled for 30–32 cycles. Real-time PCR was performed on a Bio-Rad iCycler maching with SYBR-green iQ Mix (BioRad). Standard curves were generated via amplification of 10-fold plasmid serial dilutions, and ß-actin was used for normalization. Tsix was amplified with primers oNS18 and oNS19 [13] and Xite was amplified with primers NGP3 and NGP4 [12]. Values were normalized to expression of ß-actin. Generation of Tsx targeting vector and KO mice The Tsx targeting construct was generated by PCR amplification of three segments of the Tsx gene and upstream region, verified by DNA sequencing, then cloned into a modified version of the ploxP-2FRT-PGKneo vector (a gift from Dr. David Gordon via the University of Michigan Transgenic Animal Model Core Facility) that contained a second loxP site between KpnI and BamHI. The upstream Tsx fragment (a 4.8 kB section located 6.6 kB upstream of exon 1) was cloned into the EcoRI site, the ‘middle’ fragment (1.8 kB upstream of exon 1 and 160 bp of intron 1) was cloned into the BamHI and SalI sites, and the ‘last’ fragment (intron 1 through intron 4 of Tsx) was cloned into the XhoI site. In summary, a 2.1 kB region encompassing the predicted Tsx promoter region upstream of exon 1 and 160 bp of intron 1 was flanked by loxP sites for Cre-mediated deletion of the Tsx gene. The neomycin selection marker (under control of the PGK promoter) was flanked by FRT sites, and was used for positive selection (300 µg/mL) of ES cell clones. A novel PacI site was introduced at the end of the last fragment for linearization prior to electroporation. Male TC1 ES cells (derived from 129S6/SvEvTac mice) were electroporated with 20 µg of linearized Tsx targeting construct DNA, and 400 neomycin resistant clones were picked. Genomic DNA was isolated from these clones, and digested with MscI for screening via Southern blotting using an external 1 kB probe (Tsx7) overlapping exon 5. Tsx probe 7 was generated using primers Tsx 7 F 5′-GCCTCCACTAGCACATGACA-3′ and Tsx 7 R 5′-CCCTCAGTCCTGCCTCTACC-3′. Three positive clones, containing just one integration of the targeting construct, were obtained, and clone 3A8 was selected for C57BL/6J blastocyst injection at the Brigham & Women's Hospital Transgenic Mouse Facility (Boston, MA). Two chimeric males were obtained following injection, and both transmitted the construct through the germline by matings to C57BL/6J females. Brown female pups were genotyped by Southern blot then mated to male animals expressing FLPe under control of the ROSA26 promoter (129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J; Jackson Labs) mice to remove the neomycin selection marker. Animals were genotyped by Southern blot, digesting DNA with SphI and using an internal 1 kB probe (Tsx L2), located between exons 3 and 4. Tsx probe L2 was generated using primers Tsx L2 F 5′-ACATCCCCCATGAAAACTGA-3 and Tsx L2 R 5′-ACCAAAACCAAAACCCAACA-3′. Positive animals were then mated once to C57BL/6J, pups were genotyped using SphI and probe Tsx L2, and positive animals were selected for mating with animals expressing Cre-Recombinase under control of the adenovirus EIIa promoter (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J; Jackson Labs). Following Cre-mediated deletion of Tsx, animals were genotyped by Southern blot using BstZ17I digestion and probe Tsx L2. Positive animals were then outcrossed to C57BL/6J animals for a total of 6 generations. All animals were weaned at 4 weeks, then tagged and tailed for DNA isolation for genotyping. Northern analysis of Tsx transcripts RNA (20 µg/lane) isolated from adult tissues was separated on denaturing 1% agarose-formaldehyde gels for 3 hours at 100V, then transferred to a Hybond-XL membrane overnight. The membrane was pre-hybridized using UltraHyb or buffer (Ambion) for 1 hour at 45°C, followed by overnight hybridization of the 32P-radiolabeled probe. The radiolabeled Tsx cDNA probes (both anti-sense and sense orientations consisting of Tsx exons 1–7), and the ß-actin probe were generated by linear amplification using the Ambion Strip-EZ kit and PCR primers Tsx cDNA F2 (sense strand), Tsx cDNA R2 5′-ATTGGAAGTTTGGCAAGCAA-3′ (for antisense strand), and ß-actin R. The following morning, the membranes were washed twice with low-stringency buffers (2×SSC/0.1% SDS) followed by two washes with high-stringency buffers (0.1×SSC/0.1% SDS), at 45°C. The membranes were stripped according to the kit instructions then probed for ß-actin. The membranes were visualized by exposing to a phosphoimaging screen. The male testes blot was exposed for 2 hours, and the male lung blot was exposed for 1 day. Histology, TUNEL analysis, and immunofluorescence staining Histological analysis was carried out on 4% paraformaldehyde-fixed testes that were paraffin-embedded, then sectioned at 5 µM thickness. Testis sections were deparaffinized using two changes of Histoclear (National Diagnostics) and hydrated to water by successive 2 min washes in 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and distilled H2O. Slides were then incubated in 10 mM sodium citrate pH 6.0 at 100°C for 20 min, followed by 20 min incubation at room temperature. Following unmasking, slides were washed twice in PBS. The TUNEL mixture (In Situ Cell Death Detection kit, Fluorescein; Roche) was incubated for 60 min at 37°C. The slides were washed three times in PBS for 5 min, then mounted with Vectashield mounting media containing DAPI. For immunofluorescence co-staining with SCP1 and Stra-8 antibodies, slides after TUNEL were blocked with 5% BSA in PBS-Tween-20 for 20 min at room temperature. The primary antibody was added (SCP1 at 1∶100 dilution; Stra-8 at 1∶400 dilution) and slides were incubated overnight at 37°C. The next morning the slides were washed three times in PBS-Tween-20 then incubated with a 1∶500 dilution of goat anti-rabbit Cy3 antibody for 30 min at room temperature. Slides were washed three times in PBS-Tween-20 then mounted with Vectashield containing DAPI. Tsx KO ES cell derivation and differentiation Tsx KO male and KO/KO female animals (ages 8–12 weeks) were naturally mated and females were sacrificed at 3.5dpc. Blastocysts were flushed out of the uterine horns and plated on gelatinized 15 cm plates containing mouse embryonic fibroblasts (MEFs) as described in [38]. The inner cell mass (ICM) was dissected five days later, trypsinized in a droplet, and plated onto a fresh well of MEFs. The cells were passaged and expanded until there was sufficient numbers to culture in a T25 flask (a total of 3–4 passages after ICM dissection). The Tsx KO cell lines were genotyped for gender using Zfy, NS18, and NS19 primers as described previously [39]. For differentiation experiments, ES cells were typsizinized and one million cells were plated (in triplicate) in petri dishes in ES medium lacking LIF, using the EB suspension method described previously [8]. Tsx KO cell lines of the same passage number (spanning passage number 6,7,8,9) were used for each differentiation experiment, and four independent differentiations were performed. The medium was changed every two days, and cellular cytotoxicity and viability was determined for 100,000 cells at each time point using the MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega). Cell growth of undifferentiated ES cells was determined by plating 150,000 cells per well (in triplicate) of a 12-well gelatinized plate containing MEFs. The medium was changed daily, and cells were harvested at different time points by trypsinization and counted using a Cellometer (Nexcelom Bioscience). Open field task Spontaneous locomotor activity was monitored using a MED-OFA-MS open field test system (Med Associates, St. Albans, VT). The animal was placed in the center of the activity-field arena, which is a transparent Plexiglas cage (W×D×H; 27×7×20 cm) equipped with three 16 beam infrared transmitter and sensor arrays to register horizontal and vertical activity. Ambient conditions included moderate levels of illumination and white noise (800 lux and 40 dB, respectively). The mouse's position and movement is monitored continuously in the horizontal and vertical planes by dual 16-beam infrared beam arrays. The central zone area was defined as 20×20 cm; the left arena was defined as peripheral zone. Total distance traveled, ambulatory time, ambulatory counts, stereotypy time, stereotypy counts, resting time, vertical counts, vertical time, zone entries, zone time, jump counts, jump time, average velocity, and ambulatory episodes were recorded for each test mouse throughout the 60 min. test session. Total distance provides an index of activity, while the proportion of time or distance spent in the center is taken as a measure of anxiety. Elevated plus maze The elevated plus maze (Med Associates) consists of a plus-shaped runway with two horizontal open arms and two horizontal closed arms (each 6 cm wide×35 cm long) joined by a 6 cm square center platform. The closed arms are enclosed by 20 cm black polypropylene walls. Mice are placed in the center square and allowed to explore freely under ambient light for five minutes. The number of entries and time spent in each arm is recorded. Open arm entries and occupancy provide an inverse measure of anxiety. Cued and contextual fear conditioning The fear conditioning tasks were conducted as described [40], [41]. Training session consisted of a 3 min exploration period followed by three CS-US pairings separated by 1 min (foot-shock intensity 0.8 mA, duration 0.5 s; tone 75 db white noise for 30 sec). Context tests were performed in the same training chamber after retention delays of 1 hr and 24 hr. Tone tests were performed in an environmentally altered testing chamber (different flooring and additional shelter) 24 hrs following training; baseline freezing was monitored (2 min) prior to phasic presentation of the tone (75 db white noise, 3 min duration). Baseline freezing was monitored for 2 min prior to phasic presentation of the tone (75 db white noise, 3 min duration). Mice were trained and tested in conditioning chambers that had a stainless steel grid floor through which footshocks could be delivered (Med Associates, St. Albans, VT). During training and testing sessions, the mouse's position in the chamber is recorded, digitized and analyzed using a video tracking system interfaced with a custom software package. Control and mutant groups consisted of age-matched male and female littermates (8–10 weeks of age) for each analysis. Female animals were group housed (4 animals per cage) for 2 weeks before testing in order to synchronize estrus cycles. Data are presented as group means ± SE. One way and two way ANOVA and Student t-test were used to determine statistically significant differences. For all experiments, the experimenter was blind to genotype. Supporting Information Figure S1 Open field tests and elevated plus maze tests on male TsxKO mice. (A) Open field test quantifying the time spent at the periphery (Zone R) of the chamber. (B) Elevated plus maze test quantifying the average number of entries into both open and closed areas. (C) Elevated plus maze test for the average time spent in both open and closed area. (TIF) Click here for additional data file. Figure S2 Fear conditioning test in female TsxKO mice. Contextual fear conditioning tests for 1 h and 24 h after training. The mean percentage of time spent freezing for one of two independent experiments (yielding similar results) is shown, for both wildtype (n = 9) and Tsx −/− (n = 9) female animals. (TIF) Click here for additional data file.
