小鼠大小肠表皮细胞 | 单细胞测序

 

2023年07月05日

分析单细胞数据已有5年的经验，但竟然没有从头生成过自己的数据。

小鼠基本是机制类探索项目的必备实验，不需要太复杂，只要简单的杂交取样即可。

 

小鼠模型：

Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation

这篇文章基本涵盖了CRC常见的小鼠模型

• Lgr5^eGFP; Apc^f/f;tdT (Tam induction)

• AOM/DSS
• MNU Tx
• CDX2

 

这部分是小鼠的购买和育种，并且检测是否是特定的基因型。

The generation of Lgr5eGFP-IRES-CreERT2 mouse was described earlier (69). Lgr5eGFP-IRES-CreERT2 mice were backcrossed in C57bL/6J mice and subsequently single-nucleotide polymorphism (SNP)–tested to ensure >97% pure background (Taconic). To inactivate Apc in intestinal tissue, we crossed Apcf/f mice (a gift from C. Perret) to Lgr5eGFP-IRES-CreERT2. These mice were further crossed to R26-LSL-tdTomato purchased from the Jackson Laboratory (JAX, stock #007905) (70) for conditional tdT labeling of Lgr5+ stem cells and their progeny. To delete Sox9, mice were crossed to Sox9flox mice (JAX, stock #013106) (53).
To activate conditional alleles, experimental mice aged 6 to 8 weeks were injected intraperitoneally with a single-dose tamoxifen (50 μl of sunflower oil at 10 mg/ml) unless otherwise indicated. For MNU model, Lgr5eGFP-IRES-CreERT2 mice were treated with drinking water containing 240 parts per million of MNU (bioKEMIX) scheduled every other week for 10 weeks and followed for 1 year. For AOM/DSS model, AOM/DSS treatment was performed as previously described (71). Briefly, at 8 to10 weeks of age, mice were injected intraperitoneally with AOM (10 mg/kg; Sigma-Aldrich) and treated with 2% DSS (30 to 50 kDa; colitis grade; MP Biomedicals) in drinking water for 5 days. DSS treatment was repeated three times once per 4 weeks. Mice were euthanized 11 weeks after AOM injection.

 

mice高度依赖Cre/LoxP系统来敲除指定的基因，比如Apc，或者Kras^G12D突变

• Apc需要Tam诱导30天，可以看到清晰的polyps；
• Apc+KrasG12D则只需要7天，只能看到很大的leision；

 

小鼠表皮细胞提取步骤：

1. kill mouse using CO2

2. open mouse, find cecum (connect small intestine and large colon)

3. 如果是CDX2模型，那么久collect from cecum to anus

4. open colon，see if there‘re tumors/lesions, in PBS

5. use glass slides to separate mucosa from muscle (两个载玻片即可)

6. digest to single cell using collagenase (an enzyme) for 20 mins

7. filter cells

8. cell counting

9. cell sorting (need pos and neg controls), 比如tdT，neg control就是没有Tam诱导的colon组织，需要单独准备一个mice

10. send single cells in WRN with DAPI to core for scRNA-seq

 

cell sorting

• cell size, should be single cells
• DAPI, remove dead cells
• tdT, epitheliums