二、Cre-loxP的应用
携带 loxP 的小鼠被称为 Floxed 小鼠,当它与 Cre 小鼠交配后,Cre-Lox 系统开始工作,可以对两个 loxP 位点以内的基因进行重组,进而实现基因敲除、表达等目的。
1. 组织特异性敲除(conditional knockout, cKO)
当组织特异性 Cre 小鼠(具有组织靶向性的 Cre 小鼠品系很多)与 Floxed 小鼠配繁后,两个 loxP 位点位于同一条染色体上,且方向相同。Cre 重组酶可以介导特定组织中两个同向 loxP 位点发生同源重组。致使“Gene X”处序列被删除,如果该序列的删除使得该基因发生移码突变,则达到基因敲除的效果(如图2)。
2. 组织特异性过表达(conditional knockin, cKI)
Floxed 小鼠转入某个基因的编码序列,并在这段编码序列前面插入“LoxP-STOP-LoxP, LSL”元件。因此,该Floxed小鼠并不表达插入的基因;当存在Cre重组酶时,STOP元件被删除,插入基因才会表达。该体系可用于构建基因条件性敲入小鼠模型(如图3)。
三、基因工程小鼠的一般简写方式
1.基因敲除
可以分别用加、减号上标来表示 wildtype allele 与 mutant allele:
KO 纯合子 Gene-/-
KO 杂合子 Gene+/-
野生型对照 Gene+/+ 或 WT (wildtype)
2.基因敲入
将敲入的元件写在上标里,例如:
Shh 基因 E177A 点突变杂合子:Shh E177A/+
如果 Shh 基因敲入报告基因或Cre重组酶基因,可以写成 Shh EGFP/+,或者也可以直接写成 Shh-EGFP、Shh-Cre。
3.转基因
一般直接写出表达的基因结构,例如:
Villin promoter 驱动 EGFP 报告基因的转基因小鼠就可以表示为 Vil-EGFP。
4.条件性基因敲除
将 flox 作为上标表示,比如:
CKO 纯合子 Gene flox/flox 或 Gene f/f
CKO 杂合子 Gene flox/+ 或 Gene f/+
如果与广泛表达 Cre 或生殖系表达 Cre 工具鼠交配后获得了全身敲除的小鼠,那么可以按 KO 小鼠的规则来简写。
如果与组织特异性 Cre 小鼠交配,那么可以组合写成:Gene flox/flox;Cre。
如果文章中只用到一种组织特异性 Cre 工具鼠,也可以按 KO 的方式简写,即 Gene -/- 可以表示 flox 纯合子且 Cre 阳性小鼠。
如果有多种不同的 Cre,那么就需要分别表示了。
例如:Tlr5 flox 小鼠分别与小肠上皮细胞(IEC)特异性 Cre(Vil-Cre)、 DC 细胞特异性 Cre(CD11c-Cre)交配的子代中发生基因敲除的纯合子小鼠可以分别表示为 Tlr5 flox/flox;Vil-Cre 和 Tlr5 flox/flox;CD11c-Cre。如果觉得这样写太麻烦,也可以这样表示:Tlr5 ΔIEC 和 Tlr5 ΔDC。
摆大烂
To determine if a long noncoding RNA (lncRNA) has cis-regulatory functions, several criteria are typically considered:
Proximity to Target Gene: The lncRNA should be located near the target gene on the same chromosome.
Expression Correlation: There should be a correlation between the expression of the lncRNA and the target gene.
Functional Evidence: Experimental evidence, such as loss-of-function or gain-of-function studies, should show that the lncRNA can regulate the target gene’s expression.
Physical Interaction: The lncRNA should physically interact with the target gene’s DNA or associated chromatin.
Conservation: The regulatory function of the lncRNA should be conserved across different species or cell types, if applicable.
Testing the proximity of a long noncoding RNA (lncRNA) to its target gene involves several methods:
Genomic Location Analysis: By examining the genomic coordinates of lncRNAs and potential target genes, researchers can determine physical proximity within the genome.
Expression Studies: Correlating the expression levels of lncRNAs and nearby genes can suggest regulatory relationships, especially when changes in lncRNA expression affect the gene’s expression.
Chromatin Conformation Capture (3C): Techniques like 3C or Hi-C can be used to detect physical interactions between the lncRNA and the target gene’s DNA, indicating proximity within the three-dimensional structure of the genome.
Computational Prediction: Databases like LncRNA2Target provide information on experimentally validated lncRNA-target interactions, which can be used to predict potential cis-regulatory relationships.
Experimental Validation: Techniques such as RNA immunoprecipitation or RNA pull-down assays can confirm the physical interaction between the lncRNA and the target gene’s DNA or RNA1.
These methods collectively help in establishing the cis-regulatory role of lncRNAs by confirming their proximity to target genes.
ceRNA stands for competing endogenous RNA. In molecular biology, ceRNAs are transcripts that regulate other RNA molecules by competing for shared microRNAs (miRNAs). This interaction is part of a complex regulatory network where ceRNAs can influence the levels and activity of miRNAs, thereby affecting the expression of miRNA targets.
The ceRNA network includes various types of RNAs such as long noncoding RNAs (lncRNAs), pseudogenes, and circular RNAs (circRNAs), all of which can act as molecular sponges. They bind to miRNAs through miRNA response elements (MREs), reducing the miRNAs’ ability to target other RNAs. This mechanism is crucial for maintaining the balance of gene expression and has implications in various biological processes and diseases, including cancer
The flox and Cre systems are integral parts of genetic engineering, particularly in creating genetically modified organisms like mice for research purposes. Here’s a more detailed explanation:
Flox: The term “flox” refers to a DNA sequence that has been flanked by loxP sites. These loxP sites are specific 34-base pair sequences that are recognized by the Cre recombinase enzyme1. When a gene is “floxed,” it means that it can be conditionally removed or altered by the action of Cre recombinase, which allows researchers to study the function of the gene in a controlled manner.
Cre: Cre is an enzyme called Cre recombinase that comes from bacteriophage P1. It recognizes loxP sites in the DNA and can cut and rejoin the DNA at these sites1. This allows for precise manipulation of the genome, such as the deletion, insertion, or inversion of DNA segments. The activity of Cre recombinase can be controlled so that it only acts in specific cell types or in response to certain stimuli, making it a powerful tool for studying gene function and creating models of human disease2.
The Cre-Lox system is widely used in neuroscience and other fields of biology to study complex systems and diseases. For example, it can be used to delete a gene in only certain types of neurons, which helps scientists understand the role of that gene in brain function1. It’s also used in creating models for diseases like cancer, where researchers can activate or deactivate cancer-related genes in specific tissues1.
This system provides a high level of control over gene expression and is invaluable for research that requires the study of gene function in living organisms. It’s one of the most precise tools available for genetic manipulation and has revolutionized the field of genetics.
1. 定义研究目标
- 探索 circ_0002111 在甲状腺乳头状癌(PTC)进展中的调控机制
2. 提出假设
- circ_0002111 在 PTC 中上调并影响细胞增殖、迁移和糖酵解
3. 设计实验
a. RT-qPCR 测量 circ_0002111 表达水平
b. MTT 实验检测细胞增殖
c. EdU 掺入实验检测 DNA 合成
d. 集落形成实验检测长期增殖
e. Transwell 实验检测细胞迁移
f. Seahorse 分析进行代谢剖析
g. 西方印迹法分析蛋白表达
h. 小鼠体内移植瘤实验检测体内肿瘤形成能力
i. RNA 拉下实验确定 circ_0002111 的相互作用
j. 双荧光素酶报告基因表达调控实验
4. 进行实验
- 执行上述实验并记录观察结果
5. 数据分析
- 与对照组比较结果,确定 circ_0002111 的效果
6. 得出结论
- 评估数据是否支持假设
7. 报告发现
- circ_0002111 通过海绵 miR-134-5p 和调节 FSTL1 促进 PTC 进展
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