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Jun 2014
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Wholemount in situ Hybridization for Spatial-temporal Visualization of Gene Expression in Early Post-implantation Mouse Embryos
全座原位杂交技术对植入后早期小鼠胚胎中基因表达的时空可视化   

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Abstract

Regionalized distribution of genes plays crucial roles in the formation of the spatial pattern in tissues and embryos during development. In situ hybridization has been one of the most widely used methods to screen, identify, and validate the spatial distribution of genes in tissues and embryos, due to its relative simplicity and low cost. However, acquisition of high-quality hybridization signals remains a challenge while maintaining good tissue morphology, especially for small tissues such as early post-implantation mouse embryos. In this protocol, we present a detailed RNA in situ hybridization protocol suitable for wholemount early post-implantation mouse embryos and other small tissue samples. This protocol uses digoxigenin (DIG) labeled riboprobes to hybridize with target transcripts, alkaline phosphatase-conjugated anti-DIG antibodies to recognize DIG-labeled nucleotides, and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates for color development. Specific steps and notes on riboprobe preparation, embryo collection, probe hybridization, and color development are all included in the following protocol.


Graphic abstract:



Overview of Wholemount in situ Hybridization in Early Mouse Embryos.


Keywords: Wholemount in situ hybridization (原位杂交), Mouse embryo (小鼠胚胎), Gene expression visualization (基因表达的可视化), Hybridization (杂交)

Background

Wholemount in situ hybridization has been widely used to explore gene expression distribution in both tissues and sections (Hauptmann and Gerster, 1994; Nieto et al., 1996). In the field of developmental biology, information on the spatial and temporal distribution of gene expression revealed by in situ hybridization has facilitated the identification of master regulators of embryogenesis. In our recent study, we reported that Pou3f1 is an important regulator of mouse neuroectoderm development by combining wholemount in situ hybridization and multiple functional analyses (Zhu et al., 2014). We optimized a wholemount RNA in situ hybridization protocol that uses digoxigenin labeled RNA probes and an anti-digoxigenin antibody conjugated with alkaline phosphatase to detect the enrichment of Pou3f1 in the anterior embryonic region of the mouse gastrula, which indicated potential biological functions of Pou3f1 in embryonic ectoderm development. Thereafter, more lineage regulators of the mouse gastrulation have been revealed and validated using this optimized protocol (Yang et al., 2018 and 2019; Peng et al., 2016 and 2019). The current protocol exhibits strong experimental robustness and displays application potential in a wide range of biological studies. Thus, we summarize the protocol here, in the hope its application can facilitate the study of gene expression.


The wholemount RNA in situ hybridization assay starts with the preparation of digoxigenin labeled RNA probes corresponding to target gene transcripts by using an in vitro transcription system and digoxigenin labeled dNTP mix. Pre-fixed embryo samples are treated with H2O2 and protease K for antigen retrieval and permeabilization. Embryos are then incubated with RNA probes and hybridized overnight. Several rounds of stringent wash are performed to remove unbound RNA probes. Subsequently, an antibody that recognizes digoxigenin is added to the reaction system and incubated overnight. Color development is performed to visualize the signal, and samples can be stored in a 50% Glycerol/PBS solution.


Further extensions based on the current protocol can be explored in the future, including, but not limited to, replacing digoxigenin labeled RNA probes with multiple fluorescent RNA probes, replacing the AP-conjugated DIG antibody with fluorescent conjugated antibodies, and even combining this method with protein immunofluorescence staining. However, for that to occur, essential optimization and adjustment of experimental conditions should be carefully performed. Noticeably, multiple alternative methods have been established these days, such as RNAscope (Wang, 2012). We recognize that the current protocol may exhibit a relative low detection sensitivity in comparison with RNAscope. Nevertheless, the outstanding properties of experimental robustness, no requirement for specialized instruments, and extremely low economic cost undoubtedly make our protocol an excellent option for the rapid screening and validation of gene expression in multiple fields of biological research.

Materials and Reagents

Note: All materials and reagents should be prepared in a DNase and RNase free environment unless otherwise described.

  1. Pipette tips: 10 µl, 20 µl, 200 µl, 1,000 µl Microvolume tips (Axygen®, catalog numbers: TF-300-R-S, TF-20-R-S, TF-200-R-S, TF-1000-R-S)

  2. Eppendorf tubes (Axygen®, catalog number: MCT-150-C)

  3. 35 mm × 10 mm dish (Corning, catalog number: CLS430165)

  4. 24-well plate (Corning, catalog number: 3524)

  5. Paraformaldehyde (PFA; Sigma-Aldrich, catalog number: P6148-1kg)

  6. DPBS (Gibco, catalog number: 14190144)

  7. Tween-20 (Sigma-Aldrich, catalog number: P9416-100ML)

  8. Invitrogen UltraPureTM SSC, 20× (Thermo Fisher Scientific, catalog number: 15557044)

  9. Yeast RNA (Sigma-Aldrich, catalog number: 10109223001)

  10. Heparin (Sigma-Aldrich, catalog number: H3149-500ku)

  11. RiboLock RNase Inhibitor (Thermo Fisher Scientific, catalog number: Eo0382)

  12. ScriptMAX Thermo T7 Transcription Kit (Toyobo, catalog number: TYB-TSK-101)

  13. Methanol (e.g., Sensi Chemical)

  14. Formamide (e.g., Sensi Chemical)

  15. Proteinase K solution (Invitrogen, catalog number: AM2548)

  16. Glutaraldehyde (Sinopharm Chemical, catalog number: 30092436)

  17. DIG RNA Labeling Mix (Sigma-Aldrich, catalog number: 11277073910)

  18. Anti-Digoxigenin AP antibody (Roche, catalog number: 11093274910)

  19. NBT/BCIP stock solution (Sigma-Aldrich, catalog number: 11681451001)

  20. Glycerol (Sigma-Aldrich, catalog number: G9012-100 ml)

  21. QIAquick Gel extraction kit (QIAGEN, catalog number: 28704)

  22. MEGAclearTM Kit (Ambion, catalog number: AM1908)

  23. DNase I (RNase-free) (New England Biolabs, catalog number: M0303S)

  24. 30% H2O2 (w/w) in H2O (Sigma-Aldrich, catalog number: H1009-100ML)

  25. UltraPure 0.5 M EDTA, pH 8.0 (Invitrogen, catalog number: 15575020)

  26. Albumin, Bovine Serum, Fraction V, Crystalline (Sigma-Aldrich, catalog number: 9048-46-8)

  27. NaCl (Sigma-Aldrich, catalog number: S5886-1KG)

  28. Tris base (Sigma-Aldrich, catalog number: TRIS-RO)

  29. Magnesium chloride (MgCl2; 1.00 M ± 0.01 M; Sigma-Aldrich, catalog number: M1028-100ML)

  30. KOD FX neo (Toyobo, catalog number: KFX-201)

  31. UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen, catalog number: 10977015)

  32. 4% PFA (see Recipes)

  33. PTW buffer (see Recipes)

  34. 20 mg/ml Yeast RNA (see Recipes)

  35. 50 mg/ml Heparin (see Recipes)

  36. Hybridization solution (see Recipes)

  37. 10× TBST stock (see Recipes)

  38. Blocking buffer (see Recipes)

  39. NTMT buffer (see Recipes)

  40. 4% PFA/0.1% glutaraldehyde (see Recipes)

  41. 6% H2O2/PTW solution (see Recipes)

Equipment

  1. Thermal cycler (Applied Biosystems, model: 9700)

  2. Thin-walled PCR tubes with caps (Axygen®, catalog number: PCR-02-L-C)

  3. NanoDrop 2000 (Thermal Scientific)

  4. Hybridization incubator (SciGene, model: 2000)

  5. Olympus SZX10/16 microscope

Procedure

Note: All steps should be performed in a DNase and RNase free environment unless otherwise described.

  1. Collection of Sample/Embryo

    1. Carefully collect tissue samples/mouse embryos (Figure 1) in a 35 mm dish or 24-well plate with DPBS (Downs and Davies, 1993; Piliszek et al., 2011; Pereira et al., 2011).



      Figure 1. Representative images of the unstained collected mouse gastrula at E6.5, E7.0, and E7.5 stages.

      Images were acquired with a Olympus SZX10/16 microscope. Scale bars: 100 μm.


    2. Fix embryos in 4% PFA (see Recipes) at 4°C overnight.

    3. Transfer the embryos into a graded series of methanol (25% Methanol/DPBS; 50% Methanol/DPBS; 75% Methanol/DPBS; 100% methanol) at room temperature (RT). Embryos are dehydrated for 5 min in each condition. Sufficient volume should be applied to completely submerge the embryo samples.

      Note: Prepare graded series of methanol buffer right before use.

      Pause point: The dehydrated embryos could be stored in 100% methanol at -20°C for up to 1 week.


  2. Preparation of digoxigenin labeled RNA Probes

    1. Primer design for target cDNA sequence cloning:

      For direct transcription of the PCR product in vitro, a minimal T7 promoter sequence (5-TAATACGACTCACTATAGGGAGA-3) should be added to the 5’ terminal of the primer. The length of probe sequence should be 250-1,500 bases; probes with 600-900 bases exhibit the highest sensitivity and specificity.

    2. PCR amplification and purification of probe DNA:

      A cDNA pool with high enrichment of target transcripts was used as PCR template for amplification of probe DNA. DNA polymerases such as KOD FX Neo with high fidelity characteristics are recommended. The exact PCR conditions should be adjusted according to the selected probe. After agarose gel separation, excise target DNA fragments precisely and perform gel extraction following the manufacturer’s instructions (Figure 2). Determine the concentration of acquired DNA using NanoDrop 2000.

      Note: For in vitro transcription from plasmid, the plasmid should be linearized with appropriate restriction enzyme digestion and purified with a commercial kit.



      Figure 2. Specific probe DNA (here for Tal1 gene) amplified through PCR.

      To determine PCR specificity, the PCR product is subjected to agarose gel electrophoresis. A specific DNA band can be observed, excised, and purified for further usage.


    3. Transcription of the DIG probe:

      1. Prepare the following reaction system:

        Component    For 1 µg DNA
        DNA    1 µg
        10× transcription buffer    3 µl
        DIG-nucleotide mix    2 µl
        RiboLock RNase Inhibitor    1 µl
        T7 RNA polymerase    2 µl
        Water      to 30 µl
        Total    30 µl

      2. Incubate the reaction at 37°C for 3 h in the thermocycler, with lid temperature no higher than 55°C.

      3. To remove the template DNA, add 0.5 µl DNase I to the reaction mix directly and mix well, then incubate the mix at 37°C for 15 min.

      4. Purify the acquired RNA transcript with MEGAclearTM Kit following the manufacturer’s instructions or perform phenol:chloroform extraction followed by alcohol precipitation manually. The RNA probes can be directly dissolved in nuclease free water.


  3. Sample rehydration, antigen retrieval, and permeabilization

    1. Rehydrate the embryos in graded methanol/PTW buffer (see Recipes) (75%, 50%, and 25% methanol in PTW) for 2-4 min in each concentration, allowing embryos to settle down to the bottom between changes. Wash embryos with PTW for 10 min twice.

      Note: Prepare graded series of methanol buffer right before use.

    2. Incubate the embryos in 6% H2O2/PTW solution at RT for 10 min, and then wash twice with PTW buffer.

    3. Dilute proteinase K in PTW buffer at a final concentration of 10 µg/ml proteinase K in the reaction mix. Remove PTW buffer thoroughly and incubate embryos in 10 µg/ml proteinase K reaction mix at RT. The reaction duration varies for different embryo stages. To specify, for embryos ranging from E7.0 to E9.0 embryos, the appropriate reaction duration should be 7-20 min, but longer times should be pre-tested for more advanced embryos. A pre-experiment for optimization of the conditions is strongly recommended.

    4. Remove proteinase K buffer carefully and rinse with PTW buffer twice.

    5. Post-fix the digested embryos in 4% PFA/0.1% glutaraldehyde fixation mix (see Recipes). Incubate the embryos for 20-30 min at RT.

    6. Remove the fixation buffer, and carefully wash with PTW buffer twice.


  4. Hybridization of RNA probes to the embryos

    1. Wash the embryos with hybridization solution warmed at 68°C twice; add the hybridization solution (see Recipes) and allow embryos to equilibrate until they sink to the bottom.

    2. Incubate for 2-6 h at 65-72°C. The optimal temperature varies between different RNA probes. Usually, a 68°C hybridization temperature works for most probes we have tested.

    3. Remove the hybridization solution and replace with the probe diluted in hybridization solution (200-500 ng/ml). Incubate the embryos at their corresponding temperature overnight.

    4. Re-collect the probe. Probes in hybridization solution can be re-used 6-8 times. Re-collected probes can be stored at -20°C for up to two months.

    5. Wash embryos with hybridization buffer warmed at 70°C for 30 min three times.

    6. Wash embryos with 50% hybridization buffer/50% TBST buffer at 70°C for 20 min.

    7. Wash embryos with TBST buffer (see Recipes) on a rocker platform at RT three times.


  5. Antibody incubation and digoxygenin detection

    1. Prepare blocking buffer (see Recipes) and incubate with embryos for 2-3 h at RT.

    2. Prepare antibody incubation reaction solution with 1:2,000 diluted anti-digoxigenin AP antibody in blocking buffer. Incubate on a rocker platform at 4°C overnight.

    3. Discard the antibody solution and wash the embryos with TBST buffer for 30 min three times. If required, wash the embryos with an extended overnight wash to reduce background signals.

    4. Wash the embryos twice with freshly made NTMT buffer (see Recipes).

    5. Discard the NTMT buffer and incubate embryos with NBT/BCIP solution (1:50 in NTMT buffer).

    6. Observe the signal frequently during the first two hours of NBT/BCIP solution incubation.

      Note: Refresh the NBT/BCIP solution if the solution turns red.

    7. Stop the reaction by rinsing the embryos in TBST approximately three times until an obvious signal appears.

    8. Fix the embryos in 4% PFA buffer overnight.

    9. Transfer the embryos into 50% glycerol/PBS, and record representative images of embryos samples (Figure 3).

    10. The post-fixed embryos can be store at 4°C for more than one year.



      Figure 3. Representative images of wholemount mouse early embryo in situ hybridization results of Tal1 gene.

      The images list embryos at E7.0 and E7.5 stages, from which Tal1 starts to be expressed in extraembryonic mesoderm cells, as indicated by the triangle at E7.0, and peaking at E7.5. Both embryos were stained with the same probe against the Tal1 transcript. The images were taken using an Olympus SZX10/16 microscope. Scale bars: 500 μm.

Recipes

  1. 4% PFA

    4 g of paraformaldehyde in 100 ml of DPBS, thoroughly dissolve.

    Adjust pH to 7.4-7.6 using 1 M NaOH solution and store in 4°C for up to one week.

    Note: Take care to avoid direct contact with PFA powder and solution.

  2. PTW buffer

    Calcium and magnesium free DPBS with 0.1% Tween-20.

    Store at room temperature for up to one week.

    Note: Take care to avoid direct contact with Tween-20 solution due to potential harm to skin.

  3. 20 mg/ml Yeast RNA

    Dissolve 20 mg of Yeast RNA in 1 ml of nuclease free water and mix thoroughly.

    Store at -20°C for up to one month.

  4. 50 mg/ml Heparin

    Dissolve 50 mg of Heparin in 1 ml of nuclease free water and mix thoroughly.

    Store at -20°C for up to one month.

  5. Hybridization solution

    Store at -20°C for up to one month.

    Component (stock conc.)                                      Final conc.            Volume to add

    Formamide                                                             50%                       25 ml

    SSC (20×, pH 5.3 adjusted with citric acid)            1.3× SSC                  3.25 ml

    EDTA (0.5 M, pH 8.0)                                              5 mM                    0.5 ml

    Yeast RNA (20 mg/ml in H2O)                               50 µg/ml              125 µl

    Tween-20                                                                  0.002                   100 µl

    Heparin (50 mg/ml in H2O)                                    100 µg/ml           100 µl

    UltraPureTM DNase/RNase-Free Distilled Water                                Replenish to 50 ml

    Total                                                                            50 ml

  6. 10× TBST stock

    Store at 4°C for up to one month.

    Component                           Mass

    NaCl                                       4 g

    KCl                                          0.1 g

    1 M Tris-HCl pH 7.5             12.5 ml

    Tween-20                               5.5 g

    H2O                                         Replenish to 50 ml

    Total                                        50 ml

  7. Blocking buffer

    1 mg/ml BSA in 1× TBST

    Store at 4°C for up to one week

  8. NTMT buffer

    Prepare right before use; store at room temperature for up to 2 days.

    Component (stock concentration)   Final concentration         Volume to add

    2.5 M NaCl                                           0.1 M                                             1 ml

    2 M Tris-HCl (pH 9.5)                          0.1 M                                             1.25 ml

    1 M MgCl2                                            0.05 M                                           1.25 ml

    Tween-20                                             1%                                                  0.25 ml

    H2O                                                                                                              ~21.25 ml

    Total                                                                                                              25 ml

  9. 4% PFA/0.1% glutaraldehyde

    Dilute 25% glutaraldehyde in freshly prepared 4% PFA to a final concentration of 0.1%. Prepare right before use.

    Note: Take care to avoid direct contact with PFA and glutaraldehyde solution.

  10. 6% H2O2/PTW solution

    Dilute 30% H2O2 stock buffer in freshly prepared PTW buffer to a final concentration of 6%. Prepare right before use.

    Note: Take care to avoid direct physical contact with H2O2 solution.

Acknowledgments

This work was supported in part by the National Key Basic Research and Development Program of China (2018YFA0800100, 2019YFA0801402, 2018YFA0108000, 2018YFA0107200, 2017YFA0102700), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020501 and XDA16020404), and the National Natural Science Foundation of China (31900454). This protocol was adapted from Zhu, Q., Song, L., Peng, G., Sun, N., Chen, J., Zhang, T., Sheng, N., Tang, W., Qian, C., Qiao, Y., et al. (2014). The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways. Elife 3: e02224.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Downs, K. M. and Davies, T. (1993). Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. Development 118(4): 1255-1266.
  2. Hauptmann, G. and Gerster, T. (1994). Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet 10(8): 266.
  3. Nieto, M. A., Patel, K. and Wilkinson, D. G. (1996). In: Methods in Cell Biology. Vol. 51. Bronner-Fraser, M. (Ed.). 219-235, Academic Press.
  4. Peng, G., Suo, S., Chen, J., Chen, W., Liu, C., Yu, F., Wang, R., Chen, S., Sun, N., Cui, G., et al. (2016). Spatial Transcriptome for the Molecular Annotation of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo. Dev Cell 36(6): 681-697.
  5. Peng, G., Suo, S., Cui, G., Yu, F., Wang, R., Chen, J., Chen, S., Liu, Z., Chen, G., Qian, Y., Tam, P. P. L., Han, J. J. and Jing, N. (2019). Molecular architecture of lineage allocation and tissue organization in early mouse embryo. Nature 572(7770): 528-532.
  6. Pereira, P. N., Dobreva, M. P., Graham, L., Huylebroeck, D., Lawson, K. A. and Zwijsen, A. N. (2011). Amnion formation in the mouse embryo: the single amniochorionic fold model. BMC Dev Biol 11: 48.
  7. Piliszek, A., Kwon, G. S. and Hadjantonakis, A. K. (2011). Ex utero culture and live imaging of mouse embryos. Methods Mol Biol 770: 243-257.
  8. Wang, F., Flanagan, J., Su, N., Wang, L. C., Bui, S., Nielson, A., Wu, X., Vo, H. T., Ma, X. J. and Luo, Y. (2012). RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14(1): 22-29.
  9. Yang, X., Hu, B., Hou, Y., Qiao, Y., Wang, R., Chen, Y., Qian, Y., Feng, S., Chen, J., Liu, C., et al. (2018). Silencing of developmental genes by H3K27me3 and DNA methylation reflects the discrepant plasticity of embryonic and extraembryonic lineages. Cell Res 28(5): 593-596.
  10. Yang, X., Hu, B., Liao, J., Qiao, Y., Chen, Y., Qian, Y., Feng, S., Yu, F., Dong, J., Hou, Y., et al. (2019). Distinct enhancer signatures in the mouse gastrula delineate progressive cell fate continuum during embryo development. Cell Res 29(11): 911-926.
  11. Zhu, Q., Song, L., Peng, G., Sun, N., Chen, J., Zhang, T., Sheng, N., Tang, W., Qian, C., Qiao, Y., et al. (2014). The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways. Elife 3: e02224.

简介

[摘要]基因的区域化分布在发育过程中组织和胚胎空间模式的形成中起着至关重要的作用。由于其相对简单和成本低,原位杂交已成为筛选、鉴定和验证组织和胚胎中基因空间分布的最广泛使用的方法之一。然而,在保持良好的组织形态的同时,获得高质量的杂交信号仍然是一个挑战,尤其是对于早期植入后小鼠胚胎等小组织。在这个协议中,我们提出了一个详细的 RNA原位杂交协议,适用于整装早期植入后小鼠胚胎和其他小组织样本。该协议使用地高辛 (DIG) 标记的核糖探针与目标转录本、碱性磷酸酶偶联的抗 DIG 抗体以及用于识别 DIG 标记的核苷酸和硝基蓝四唑 (NBT)/5-溴-4-氯-3-吲哚基磷酸酯杂交(BCIP) 用于显色的显色底物。riboprobe 制备、胚胎收集、探针杂交和颜色开发的具体步骤和注意事项都包含在以下协议中。

[背景] Wholemount原位杂交已广泛用于探索组织和切片中的基因表达分布(Hauptmann 和 Gerster,1994 年;Nieto等人,1996 年)。在发育生物学领域,原位杂交揭示的基因表达的时空分布信息促进了胚胎发生主要调节因子的鉴定。在我们最近的研究中,我们报道了Pou3f1是小鼠神经外胚层发育的重要调节因子,通过将整装原位杂交和多功能分析相结合(Zhu等,2014)。我们优化了全量 RNA原位杂交方案,该方案使用地高辛标记的 RNA 探针和结合碱性磷酸酶的抗地高辛抗体检测小鼠原肠胚前区Pou3f1的富集,这表明Pou3f1在胚胎中的潜在生物学功能外胚层发育。此后,使用此优化方案揭示并验证了更多小鼠原肠胚形成的谱系调节因子(Yang等人,2018 年和 2019 年;Peng等人,2016 年和 2019 年)。目前的协议表现出很强的实验鲁棒性,并在广泛的生物学研究中显示出应用潜力。因此,我们在这里总结了该协议,希望其应用能够促进基因表达的研究。

Wholemount RNA原位杂交分析从使用体外转录系统和地高辛标记的 dNTP 混合物制备与靶基因转录物相对应的地高辛标记 RNA 探针开始。预先固定的胚胎样品用 H 2 O 2和蛋白酶 K处理以进行抗原修复和透化。然后将胚胎与 RNA 探针一起孵育并杂交过夜。执行几轮严格洗涤以去除未结合的 RNA 探针。随后,将识别地高辛的抗体添加到反应系统中并孵育过夜。进行显色以显示信号,样品可以储存在 50% 甘油/PBS 溶液中。

未来可以探索基于当前协议的进一步扩展,包括但不限于用多个荧光 RNA 探针代替地高辛标记的 RNA 探针,用荧光偶联抗体代替 AP 偶联的 DIG 抗体,甚至将此方法与蛋白质免疫荧光染色。然而,为了发生这种情况,应仔细进行实验条件的基本优化和调整。值得注意的是,如今已经建立了多种替代方法,例如 RNAscope (Wang, 2012)。我们认识到,与 RNAscope 相比,当前的协议可能表现出相对较低的检测灵敏度。尽管如此,实验稳健性的突出特性,不需要专门的仪器,以及极低的经济成本,无疑使我们的协议成为生物研究多个领域中基因表达快速筛选和验证的绝佳选择。

关键字:原位杂交, 小鼠胚胎, 基因表达的可视化, 杂交

材料和试剂

 

注意:除非另有说明,所有材料和试剂均应在无 DNase RNase 的环境中制备。

1.     移液器吸头:10 µl20 µl200 µl1,000 µl 微量吸头(Axygen ® ,目录号:TF-300-RSTF-20-RSTF-200-RSTF-1000-RS

2.     Eppendorf 管(Axygen ® ,目录号:MCT-150-C

3.     35 mm × 10 mm 碟(康宁,目录号:CLS430165

4.     24 孔板(Corning,目录号:3524

5.     多聚甲醛(PFASigma-Aldrich,目录号:P6148-1kg

6.     DPBSGibco,目录号:14190144

7.     Tween-20Sigma-Aldrich,目录号:P9416-100ML

8.     Invitrogen UltraPure TM SSC20 ×Thermo Fisher Scientific,目录号:15557044

9.     酵母 RNASigma-Aldrich,目录号:10109223001

10.  肝素(Sigma-Aldrich,目录号:H3149-500ku

11.  RiboLock RNase InhibitorThermo Fisher Scientific,目录号:Eo0382

12.  ScriptMAX Thermo T7 转录套件(Toyobo,目录号:TYB-TSK-101

13.  甲醇(例如., Sensi Chemical

14.  甲酰胺(例如., Sensi Chemical

15.  蛋白酶 K 溶液(Invitrogen,目录号:AM2548

16.  戊二醛(国药集团,目录号:30092436

17.  DIG RNA 标记混合物(Sigma-Aldrich,目录号:11277073910

18.  抗地高辛AP抗体(Roche ,目录号:11093274910

19.  NBT/BCIP 原液(Sigma-Aldrich,目录号:11681451001

20.  甘油(Sigma-Aldrich ,目录号:G9012-100 ml

21.  QIAquick Gel提取试剂盒(QIAGEN ,目录号:28704

22.  MEGAclear TM套件(Ambion ,目录号:AM1908

23.  DNase I(无RNase)(New England Biolabs,目录号:M0303S

24.  30% H (w/w) in H Sigma-Aldrich ,目录号H1009-100ML

25.  UltraPure 0.5 M EDTApH 8.0Invitrogen,目录号:15575020

26.  白蛋白,牛血清,分数 V,结晶(Sigma-Aldrich,目录号:9048-46-8

27.  NaCl Sigma-Aldrich ,目录号:S5886-1KG

28.  Tris Sigma-Aldrich ,目录号:TRIS-RO

29.  氯化镁(MgCl 1.00 M ± 0.01 MSigma-Aldrich,目录号:M1028-100ML

30.  KOD FX neo Toyobo ,目录号:KFX-201

31.  UltraPure TM DNase/RNase-Free 蒸馏水(Invitrogen,目录号:10977015

32.  4% PFA(见配方)

33.  PTW 缓冲液(见配方)

34.  20 mg/ml 酵母 RNA(见配方)

35.  50 mg/ml 肝素(见食谱)

36.  杂交溶液(见配方)

37.  10× TBST 库存(见食谱)

38.  阻塞缓冲区(见食谱)

39.  NTMT 缓冲液(见配方)

40.  4% PFA/0.1% 戊二醛(见配方)

41.  6% H /PTW 溶液(见配方)

 

设备

 

1.     热循环仪(Applied Biosystems,型号:9700

2.     带盖的薄壁 PCR 管(Axygen ® ,目录号:PCR-02-LC

3.     NanoDrop 2000 (Thermal Scientific)

4.     杂交培养箱(SciGene,型号:2000

5.     奥林巴斯 SZX10/16 显微镜

 

 

程序

 

注意:除非另有说明,否则所有步骤都应在无 DNase RNase 的环境中执行。

A.    样本/胚胎的收集

1.     DPBS 35 毫米培养皿或 24 孔板中小心地收集组织样本/小鼠胚胎(图 1)(Downs Davies1993 年;Piliszek等人2011 年;Pereira等人2011 年)。

 

 

1. E6.5E7.0 E7.5 阶段收集的未染色小鼠原肠的代表性图像。

图像是用 Olympus SZX10/16 显微镜采集的。比例尺:100μ米。

 

2.     °C 过夜固定 4% PFA(见食谱)中的胚胎

3.     在室温 (RT) 下将胚胎转移到分级系列的甲醇中 (25% 甲醇/DPBS;50% 甲醇/DPBS;75% 甲醇/DPBS;100% 甲醇)。胚胎在每种条件下脱水 5 分钟。应施加足够的体积以完全淹没胚胎样品。

注意:在使用前准备分级系列的甲醇缓冲液。

暂停点:脱水的胚胎可以 -20°C 100% 甲醇中储存长达 1 周。

 

B.    地高辛标记的 RNA 探针的制备

1.     目标cDNA序列克隆的引物设计:

为了在体外直接转录 PCR 产物应在引物的 5' 末端添加一个最小的 T7 启动子序列 (5-TAATACGACTCACTATAGGGAGA-3)。探针序列长度为250-1500个碱基;600-900 个碱基的探针表现出最高的灵敏度和特异性。

2.     探针DNAPCR扩增和纯化:

具有高度富集的目标转录本的 cDNA 库被用作 PCR 模板,用于扩增探针 DNA。推荐使用具有高保真特性的 DNA 聚合酶,例如 KOD FX Neo。应根据所选探针调整确切的 PCR 条件。琼脂糖凝胶分离后,精确切除目标 DNA 片段并按照制造商的说明进行凝胶提取(图 2)。使用 NanoDrop 2000 确定获得的 DNA 的浓度。

注意:对于质粒的体外转录,应使用适当的限制酶消化将质粒线性化,并使用商业试剂盒进行纯化。

 

 

2.通过 PCR 扩增的特定探针 DNA(此处为Tal1基因)。

为了确定 PCR 特异性,PCR 产物需要进行琼脂糖凝胶电泳。可以观察、切除和纯化特定的 DNA 带以供进一步使用。

 

3.     DIG 探针的转录:

a. 准备以下反应体系:

成分

对于 1 µg DNA

脱氧核糖核酸

1微克

10×转录缓冲液

3微升

DIG-核苷酸混合物

2微升

RiboLock RNase 抑制剂

1 微升

T7 RNA聚合酶

2微升

30 微升

全部的

30 微升

b. 在热循环仪中在 37°C 下孵育反应 3 小时,盖子温度不高于 55°C

c.  要去除模板 DNA,直接将 0.5 µl DNase I 添加到反应混合物中并充分混合,然后将混合物在 37°C 下孵育 15 分钟。

d. 按照制造商的说明使用 MEGAclear TM Kit纯化获得的 RNA 转录物,或执行苯酚:氯仿提取,然后手动进行酒精沉淀。RNA 探针可以直接溶解在无核酸酶的水中。

 

 

C.    样品再水化、抗原修复和透化

1.     在分级甲醇/PTW 缓冲液(参见配方)(PTW 中的 75%50% 25% 甲醇)中对胚胎进行再水化,每个浓度为 2-4 分钟,使胚胎在变化之间沉降到底部。 PTW 清洗胚胎 10 分钟两次。

注意:在使用前准备分级系列的甲醇缓冲液。

2.     RT 6% H /PTW 溶液中孵育胚胎10 分钟,然后用 PTW 缓冲液清洗两次。

3.     PTW 缓冲液中稀释蛋白酶 K,使反应混合物中的蛋白酶 K 终浓度为 10 µg/ml。彻底去除 PTW 缓冲液,并在室温下在 10 µg/ml 蛋白酶 K 反应混合物中孵育胚胎。不同胚胎阶段的反应持续时间不同。具体来说,对于从 E7.0 E9.0 的胚胎,适当的反应持续时间应为 7-20 分钟,但对于更高级的胚胎,应预先测试更长的时间。强烈建议进行条件优化的预实验。

4.     小心取出蛋白酶 K 缓冲液并用 PTW 缓冲液冲洗两次。

5.     4% PFA/0.1% 中对消化的胚胎进行后修复 戊二醛固定组合(见食谱)。在 RT 中孵化胚胎 20-30 分钟。

6.     取出固定缓冲液,用 PTW 缓冲液仔细清洗两次。

 

D.    RNA探针与胚胎的杂交

1.     68°C加热的杂交液洗涤胚胎两次;添加杂交溶液(参见配方)并让胚胎平衡直到它们沉入底部。

2.     65-72°C 下孵育 2-6 小时。最佳温度因不同的 RNA 探针而异。通常,68°C 杂交温度适用于我们测试过的大多数探针。

3.     取出杂交溶液并用在杂交溶液 (200-500 ng/ ml ) 中稀释的探针代替。在相应的温度下孵化胚胎过夜。

4.     重新收集探针。杂交溶液中的探针可重复使用 6-8 次。重新收集的探针可以在 -20°C 下储存长达两个月。

5.     用在 70°C 加热 30 分钟的杂交缓冲液洗涤胚胎 3 次。

6.     50% 杂交缓冲液/50% TBST 缓冲液在 70°C 下洗涤胚胎 20 分钟。

7.     RT 的摇臂平台上用 TBST 缓冲液(参见食谱)清洗胚胎 3 次。

 

E.    抗体孵育和地高辛检测

1.     准备封闭缓冲液(参见食谱)并在室温下与胚胎一起孵育 2-3 小时。

2.     在封闭缓冲液中用 1:2,000 稀释的抗地高辛 AP 抗体制备抗体孵育反应溶液。在摇杆平台上于 4°C 孵育过夜。

3.     弃去抗体溶液,用 TBST 缓冲液清洗胚胎 30 分钟 3 次。如果需要,用延长的隔夜洗涤时间洗涤胚胎以减少背景信号。

4.     用新鲜制作的 NTMT 缓冲液清洗胚胎两次(参见食谱)。

5.     丢弃 NTMT 缓冲液并用 NBT/BCIP 溶液(NTMT 缓冲液中的 1:50)孵育胚胎。

6.     NBT/BCIP 溶液孵育的前两个小时内经常观察信号。

注意:如果溶液变成红色,请刷新 NBT/BCIP 溶液。

7.     通过在 TBST 中冲洗胚胎大约 3 次来停止反应,直到出现明显的信号。

8.     将胚胎固定在 4% PFA 缓冲液中过夜。

9.     将胚胎转移到 50% 甘油/PBS 中,并记录胚胎样本的代表性图像(图 3)。

10.  固定后的胚胎可以在°C下储存一年以上。

 

SHAPE \* MERGEFORMAT

3. Wholemount 小鼠早期胚胎Tal1基因原位杂交结果的代表性图像。

图像列表胚胎E7.0E7.5阶段,从其中TAL1开始在胚外中胚层细胞中表达,通过在E7.0三角形所指示的,并且在E7.5峰值。两个胚胎都用相同的探针针对Tal1转录物染色。图像是使用 Olympus SZX10/16 显微镜拍摄的。比例尺:500 μm

 

食谱

 

1.     4% 煤灰

4 克多聚甲醛在 100 毫升 DPBS 中,彻底溶解。

使用 1 M NaOH 溶液将 pH 值调节至 7.4-7.6,并在°C 储存长达一周。

注意:注意避免直接接触 PFA 粉末和溶液。

2.     PTW 缓冲区

不含钙和镁的 DPBS,含 0.1% Tween-20

在室温下最多可存放 1 周。

注意:注意避免直接接触 Tween-20 溶液,以免对皮肤造成潜在伤害。

3.     20 毫克/毫升酵母 RNA

20 mg 酵母 RNA 溶解在 1 ml 无核酸酶水中并充分混合。

-20 °C 储存长达 1 个月。

4.     50 毫克/毫升肝素

50 mg 肝素溶解在 1 ml 无核酸酶水中并充分混合。

-20 °C 储存长达 1 个月。

5.     杂交方案

-20 °C 储存长达 1 个月。

组件(库存浓度) 最终浓度 要添加的音量

甲酰胺 50% 25毫升

SSC20×,用柠檬酸调节 pH 5.31.3× SSC 3.25 ml          

EDTA0.5 MpH 8.0 5毫米 0.5毫升

酵母 RNA(在O 中为20 mg/ml  50 微克/毫升 125 微升

吐温 20 0.002 100 微升

肝素(50 mg/ml O 100 微克/毫升 100 微升

UltraPure TM DNase/RNase-Free 蒸馏水 补充至 50 毫升

全部的 50毫升

6.     10× TBST 库存

°C 储存长达 1 个月。

成分 大量的

氯化钠 4

氯化钾 0.1

1 M Tris-HCl pH 7.5 12.5 毫升

吐温 20 5.5

O 补充至 50 毫升

全部的 50毫升

7.     阻塞缓冲区

1 毫克/毫升 BSA 1× TBST

°C 下最多可储存1

8.     NTMT缓冲区

使用前准备;在室温下最多可存放 2 天。

成分(库存浓度) 终浓度加入体积                                                                          

2.5 M 氯化钠 0.1M       1毫升

2 M Tris-HCl (pH 9.5) 0.1M       1.25 毫升

1 M 氯化镁2 0.05        1.25 毫升

吐温 20 1%       0.25 毫升

O       ~21.25 毫升

全部的        25毫升

9.     4% PFA/0.1% 戊二醛

在新鲜制备的 4% PFA 中稀释 25% 戊二醛至最终浓度为 0.1%。使用前准备好。

注意:注意避免直接接触 PFA 和戊二醛溶液。

10.  6% H /PTW 溶液

在新制备的 PTW 缓冲液中稀释 30% H 2库存缓冲液至最终浓度为 6%。使用前准备好。

注意:注意避免与2溶液直接物理接触。

 

致谢

 

这项工作得到了中国国家重点基础研究发展计划(2018YFA08001002019YFA08014022018YFA01080002018YFA01072002017YFA0102700)、国家自然科学基金战略研究计划(X0104000000)和国家战略研究计划(X010040002700)和国家战略研究计划(X0104002700)的部分支持。中国科学基金(31900454)。该协议改编自 Zhu, Q., Song, L., Peng, G., Sun, N., Chen, J., Zhang, T., Sheng, N., Tang, W., Qian, C.,乔,Y.(2014)。转录因子 Pou3f1 通过激活神经谱系基因和抑制外部信号通路促进神经命运承诺。Elife 3e02224

 

利益争夺

 

作者声明没有利益冲突或竞争利益。

 

参考

 

1.     Downs, KM Davies, T. (1993)通过解剖显微镜中的形态标志对原肠胚小鼠胚胎进行分期。发展118(4)1255-1266

2.     Hauptmann, G. Gerster, T. (1994)与脊椎动物和果蝇胚胎的双色整体原位杂交。趋势基因10(8): 266

3.     Nieto, MAPatel, K. Wilkinson, DG (1996)。在:细胞生物学方法。卷。51. Bronner-Fraser, M.(主编)。219-235,学术出版社。

4.     Peng, G., Suo, S., Chen, J., Chen, W., Liu, C., Yu, F., Wang, R., Chen, S., Sun, N., Cui, G.,等。(2016)用于中原肠小鼠胚胎谱系命运和细胞身份分子注释的空间转录组。开发单元36(6)681-697

5.     Peng, G., Suo, S., Cui, G., Yu, F., Wang, R., Chen, J., Chen, S., Liu, Z., Chen, G., Qian, Y., Tam, PPL, Han, JJ Jing, N. (2019)早期小鼠胚胎中谱系分配和组织组织的分子结构。自然572(7770)528-532

6.     Pereira, PN, Dobreva, MP, Graham, L., Huylebroeck, D., Lawson, KA Zwijsen, AN (2011)小鼠胚胎中的羊膜形成:单羊膜褶皱模型。BMC 开发生物学 1148 

7.     Piliszek, A.Kwon, GS Hadjantonakis, AK (2011)小鼠胚胎的体外培养和活体成像。方法 Mol Biol 770243-257

8.     Wang, F., Flanagan, J., Su, N., Wang, LC, Bui, S., Nielson, A., Wu, X., Vo, HT, Ma, XJ Luo, Y. (2012)RNAscope:一种用于福尔马林固定、石蜡包埋组织的新型原位RNA 分析平台。J Mol Diagn 141):22-29

9.     Yang, X., Hu, B., Hou, Y., Qiao, Y., Wang, R., Chen, Y., Qian, Y., Feng, S., Chen, J., Liu, C.,等。(2018)H3K27me3 DNA 甲基化对发育基因的沉默反映了胚胎和胚胎外谱系的差异可塑性。细胞研究28(5): 593-596

10.  Yang, X., Hu, B., Liao, J., Qiao, Y., Chen, Y., Qian, Y., Feng, S., Yu, F., Dong, J., Hou, Y.,等。(2019)小鼠原肠中不同的增强子特征描绘了胚胎发育过程中渐进的细胞命运连续体细胞研究29(11)911-926

11.  Zhu, Q., Song, L., Peng, G., Sun, N., Chen, J., Zhang, T., Sheng, N., Tang, W., Qian, C., Qiao, Y.,等。(2014)转录因子 Pou3f1 通过激活神经谱系基因和抑制外部信号通路促进神经命运承诺。Elife 3e02224

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Copyright Yang et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yang, X., Chen, Y., Song, L., Zhang, T. and Jing, N. (2021). Wholemount in situ Hybridization for Spatial-temporal Visualization of Gene Expression in Early Post-implantation Mouse Embryos. Bio-protocol 11(22): e4229. DOI: 10.21769/BioProtoc.4229.
  2. Zhu, Q., Song, L., Peng, G., Sun, N., Chen, J., Zhang, T., Sheng, N., Tang, W., Qian, C., Qiao, Y., et al. (2014). The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways. Elife 3: e02224.
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