There were errors published in Development138, .
The authors of this article informed us of errors with the display of data in Fig. 3 and Fig. S2 in this paper. These are detailed below, and the original data provided by the authors are also included as part of this Correction. The authors apologise to the readers for these errors. The editors of the journal have examined this paper in detail and find that, while there were substantive errors in experimental layout and data presentation in Fig. 3 and Fig. S2, these do not affect the conclusions of the paper.
PCR-RFLP analysis of the Xi pattern of CBMS1 Cdx2ER and Gata6GR cell lines. PCR fragments were cut by specific restriction enzyme sets (see Table S2 in the supplementary material). Note that two different sizes of fragments were identified in induced extra-embryonic cells, indicating the occurrence of random X inactivation in these cells. RT–, no reverse transcriptase control; TE, trophectoderm; PrE, primitive endoderm. CBA, Mus musculus musculus; MsM, Mus musculus molossinus (see Additional Materials and Methods in this Correction). The size differences between PCR fragments were analyzed in either polyacrylamide gels (a) or agarose gels (b). White lines between lanes indicate samples that were processed together but run on separate gels; band intensities are therefore not quantitatively comparable in these cases.
PCR-RFLP analysis of the Xi pattern of CBMS1 Cdx2ER and Gata6GR cell lines. PCR fragments were cut by specific restriction enzyme sets (see Table S2 in the supplementary material). Note that two different sizes of fragments were identified in induced extra-embryonic cells, indicating the occurrence of random X inactivation in these cells. RT–, no reverse transcriptase control; TE, trophectoderm; PrE, primitive endoderm. CBA, Mus musculus musculus; MsM, Mus musculus molossinus (see Additional Materials and Methods in this Correction). The size differences between PCR fragments were analyzed in either polyacrylamide gels (a) or agarose gels (b). White lines between lanes indicate samples that were processed together but run on separate gels; band intensities are therefore not quantitatively comparable in these cases.
Random Xi in Flk1+ mesodermal cells. CBMS1 ES cells were cultured on a collagen type IV-coated dish for 5 days to induce mesoderm, and Flk1+ mesodermal cells were stained by APC-conjugated anti-Flk1 antibody and separated by FACS sorting. Xi states were analyzed using the PCR-RFLP method. Two different sizes of fragments were detected by agarose electrophoresis in both ES cells and Flk1+ mosodermal cells, indicating the transcripts from both X chromosomes were present in these samples. The two bands in ES cells indicate the state of two active X chromosomes, whereas those in Flk1+ cells indicate a state of random Xi, since the uniform Xi event is confirmed by H3K27me3 staining (see Fig. S1). White lines between lanes indicate samples that were processed together but run on separate gels; band intensities are therefore not quantitatively comparable in these cases.
Random Xi in Flk1+ mesodermal cells. CBMS1 ES cells were cultured on a collagen type IV-coated dish for 5 days to induce mesoderm, and Flk1+ mesodermal cells were stained by APC-conjugated anti-Flk1 antibody and separated by FACS sorting. Xi states were analyzed using the PCR-RFLP method. Two different sizes of fragments were detected by agarose electrophoresis in both ES cells and Flk1+ mosodermal cells, indicating the transcripts from both X chromosomes were present in these samples. The two bands in ES cells indicate the state of two active X chromosomes, whereas those in Flk1+ cells indicate a state of random Xi, since the uniform Xi event is confirmed by H3K27me3 staining (see Fig. S1). White lines between lanes indicate samples that were processed together but run on separate gels; band intensities are therefore not quantitatively comparable in these cases.
In Fig. 3, most samples were analysed on polyacrylamide gels, but the G6pd and Pdha1 samples were analysed on agarose gels. The difference in experimental procedure was not stated. In addition, the ‘–RT’, ‘non-cut’ and ‘cut’ samples for these two loci were run on separate gels, although all were processed in parallel. Lanes were spliced together without showing or stating that they had come from separate gels. In these cases, it should be noted that bands are not quantitatively comparable. The corrected version of Fig. 3 and its legend are shown below. The original data for all samples in this figure are provided in Fig. A.
In Fig. S2, the ‘non cut’ and ‘cut’ samples for the CBMS1 ES lanes were run on separate gels, although processed in parallel. This was not appropriately marked on the figure. For all gels run for this figure, short and long exposure pictures were taken, and the short exposure used for assembling the original figure. Upon examination of the long-exposure pictures, it became apparent that the short exposures did not in all cases clearly show the cut bands. In the corrected version of the figure below, the long exposure has been used where appropriate. It should be noted that, since the CBMS1 ES lanes were run on separate gels, these bands are not quantitatively comparable. The authors have been unable to locate the original data for the G6pd samples, and have therefore removed these lanes from the corrected version of the figure. The original data for all other samples are provided in Fig. B. The version of Fig. S2 that appears online is the original figure without the lanes removed.
Finally, the authors omitted key parts of the Materials and Methods in the published version of the paper. The missing sections are provided here.
ADDITIONAL MATERIALS AND METHODS
CBMS1 and B142bgeoEG mESC derivation and culture condition
A hybrid F1 CBMS1 female ES cell line was derived from an embryo obtained by mating female Mus musculus musculus (CBA) and male Mus musculus molocimus (MsM) mice. A B142bgeoEG female ES cell line was derived from an embryo obtained by mating genetically modified Mus musculus (DBA) mice. Both female ES cell lines were cultured in GMEM (Gibco), 14% KSR (Gibco), 1% FCS (Thermo Scientific), 1× sodium pyruvate (Gibco), 1× NEAA (Gibco), 10–4 M 2-mercaptoethanol (Nakarai Tesque), 1000 U/ml of leukemia inhibitory factor (LIF) on a 0.1% (w/v) gelatin-coated dish.
Derivation of B142bgeoEG Cdx2ER, Gata6GR; CBMS1 Cdx2ER and Gata6GR mESC lines
A Gata6GR or Cdx2ER expression vector (50 µg) with puromycin-resistant gene (pPyCAG-Gata6GR-IP or pPyCAG-Cdx2ER-IP) (Niwa et al., 2005; Shimosato et al., 2007) was linearized and transfected into 1×107 CBMS1 ES cells by electroporation followed by the selection with 1.5 µg/ml of puromycin (Nakarai Tesque). A Gata6GR or Cdx2ER-expression vector (50 µg) with hygromycin-resistant gene (pPyCAG-Gata6GR-IH or pPyCAG-Cdx2ER-IH) was linearized and transfected into 1×107 B142bgeoEG ES cells by electroporation followed by selection with 100 µg/ml Hygromycin B (Invitrogen). After the selection for 7 days, the drug-resistant colonies were isolated and the cell lines that undergo differentiation to extra-embryonic lineage-like cells in the presence of 1 µg/ml 4-hydroxy tamoxifen (Tx) (Sigma) for Cdx2ER or in the presence of 100 nM dexamethasone (Dex) (Sigma) for Gata6GR were selected for further analyses.
Induction of trophectodermal (TE) and primitive endodermal (PrE) cells
1×106 mES cells, which express Gata6GR or Cdx2ER were seeded on a gelatin-coated dishes in GMEM, 10% FCS (Hyclone), 1× sodium pyruvate, 1× NEAA, 10-4 M 2-mercaptoethanol without LIF. After attaching the cells to the dish bottom, Tx or Dex were added and the cells were cultured for 5 days to obtain PrE and TE cells. Induction of PrE and TE were confirmed by the expression of lineage-specific markers.
Induction of Flk1+ mesodermal cells
Flk1-positive mesodermal cells were induced according to Nishikawa et al. (Nishikawa et al., 1998). Briefly, 1×104 CBMS1 and B142bgeoEG ES cells were seed onto collagen type IV-coated dishes (Nitta Gelatin) in the Flk1+ induction medium (α-MEM, 10% FCS, 10–4 M 2-mercaptoethanol, 1× NEAA). After 5 days in culture, differentiated cells were dissociated using cell-dissociation buffer (Gibco) and stained using APC-conjugated anti-mouse Flk1 antibody (eBioscience, 17-5821-80). The population of Flk1+ mesodermal cells was analyzed and sorted by FACS Aria (BD).
PCR-RFLP analysis
Total RNA was extracted from 1×106 mES, TE, PrE and Flk1+ cells using RNA extraction kit (Kurabo), according to the manufacturer's instructions. Total RNA (1 µg) was used for cDNA synthesis with ReverTra ace-alpha cDNA Synthesis Kit (Toyobo) (20 µl/1 reaction). First-strand cDNA was synthesized using the random oligo primer. RT- samples were prepared at this step in the reaction without reverse transcriptase.
RT-PCR primers were designed to detect polymorphisms that were contained X-linked gene transcripts (Sugimoto and Abe, 2007; Table S2). RT-PCR was performed using 1 µl of cDNA or RT– samples by using Taq-Gold PCR polymerase (Applied Biosystems) (20 µl/1 reaction ×5). The PCR conditions were 94°C for 30 s, 52°C for 30 s, 72°C for 30 s; 35-40 cycles (for G6pd, the annealing temperature was 50°C). PCR products were purified by phenol-chloroform treatment followed by ethanol precipitation. Finally, the PCR products were dissolved in 50 µl of TE (pH 8.0). The concentration was measured using a Nanodrop ND-1000 (Thermo Fisher Scientific).
PCR products (1 µg) were digested overnight with the restriction enzymes listed in Table S2 (20 µl/1 reaction). RT– and 1 µg of non-cut and cut samples were loaded on each well and the sizes of the DNA fragments were analyzed by electrophoresis using 2% agarose gel and 10% polyacrylamide gel.
