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。
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
[摘自公众号文章]
因此这种模式来预测靶基因的原理就是预测lncRNA序列与mRNA序列结合的自由能,自由能越小,越容易结合。
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
Here’s a summary of the key experiments and their conclusions from the article:
Circ_0002111 Expression Analysis: The study found that circ_0002111 was significantly upregulated in PTC tissues and cells, suggesting its role in PTC progression1.
Functional Assays: Silencing circ_0002111 inhibited PTC cell proliferation, migration, and glycolytic metabolism, indicating its oncogenic function23.
In Vivo Tumorigenesis: Knockdown of circ_0002111 reduced tumor growth in a xenograft model, further confirming its role in promoting PTC4.
miR-134-5p Interaction: Circ_0002111 was shown to act as a sponge for miR-134-5p, affecting its expression and PTC cell behavior.
FSTL1 Regulation: The study demonstrated that miR-134-5p targets FSTL1, and circ_0002111 can regulate FSTL1 levels by sponging miR-134-5p.
反转录定量聚合酶链反应 (RT-qPCR)
用于检测circ_0002111、miR-134-5p和FSTL1的水平。1
通过SYBR™ Green Master Mix和TaqMan™ Fast Advanced Master Mix进行定量反应。
细胞增殖评估
使用MTT试剂、EdU试剂和克隆形成试验来评估细胞增殖。
通过显微镜观察和图像分析软件计数。
细胞迁移能力测定
通过Transwell试验来确定细胞迁移能力。2
使用结晶紫染色并通过倒置显微镜观察。
糖酵解分析
通过外部酸化率(ECAR)、氧消耗率(OCR)、葡萄糖消耗和乳酸产生来分析糖酵解。
使用Seahorse XFe 96 Extracellular Flux Analyzer进行检测。
1. Define Research Objective
- Explore regulatory mechanism of circ_0002111 in PTC progression
2. Formulate Hypothesis
- circ_0002111 upregulates in PTC and affects cell proliferation, migration, and glycolysis
3. Design Experiments
a. RT-qPCR to measure circ_0002111 expression levels
b. MTT assay for cell proliferation
c. EdU incorporation assay for DNA synthesis
d. Colony formation assay for long-term proliferation
e. Transwell assay for cell migration
f. Seahorse analysis for metabolic profiling
g. Western blot for protein expression analysis
h. Xenograft tumor assay in mice for in vivo tumorigenicity
i. RNA pull-down to identify circ_0002111 interactions
j. Dual-luciferase reporter assay for gene expression regulation
4. Conduct Experiments
- Perform the above assays and record observations
5. Analyze Data
- Compare results with control groups to determine the effect of circ_0002111
6. Draw Conclusions
- Assess whether the data supports the hypothesis
7. Report Findings
- circ_0002111 promotes PTC progression by sponging miR-134-5p and regulating FSTL1
TPC1 and IHH4 are cell lines used in scientific research, particularly in the study of thyroid cancer.
TPC1 is a cell line that has the RET/PTC rearrangement, which is associated with papillary thyroid cancer (PTC). This genetic alteration is often studied because it can provide insights into the development and progression of PTC1.
IHH4 is another cell line that is mutated for BRAFV600E, also used in the context of PTC research. The BRAFV600E mutation is one of the most common mutations found in PTC and is a significant area of focus for understanding the disease and developing targeted therapies1.
The article details several experiments conducted to explore the role of circular RNA (circ_0002111) in the progression of papillary thyroid carcinoma (PTC). Here’s a detailed one-to-one list of the experiments and their demonstrations:
RT-qPCR: Used to detect the levels of circ_0002111, miR-134-5p, and FSTL1, demonstrating that circ_0002111 is upregulated in PTC samples and cells1.
Cell Proliferation Assays (MTT, EdU, Colony Formation): Showed that downregulation of circ_0002111 suppresses PTC cell proliferation.
Transwell Assay: Indicated that circ_0002111 knockdown reduces PTC cell migration ability2.
Glycolysis Analysis (ECAR, OCR, Glucose Consumption, Lactate Production): Demonstrated that circ_0002111 downregulation leads to decreased glycolytic metabolism in PTC cells.
Western Blot: Confirmed that silencing circ_0002111 reduces the levels of proteins associated with proliferation, migration, and glycolysis.
Xenograft Tumor Assay: Revealed that circ_0002111 promotes tumorigenesis in vivo34.
Dual-Luciferase Reporter and RNA Pull-Down Assays: Validated that circ_0002111 acts as a sponge for miR-134-5p and regulates FSTL1 expression.
The term “PCD5-ciR” refers to a fifth-generation circular RNA (circRNA) expression vector. It is designed with a specialized circRNA expression framework that includes carefully modified Alu elements, binding sites for RNA-binding proteins (RBPs) like QKI, and a newly designed circularization mediating sequence to ensure accurate and efficient circularization of the inserted circRNA. The expression framework has reserved EcoRI and BamHI restriction sites for direct enzyme digestion and insertion of the target circRNA fragment1.
This vector is used in molecular biology and genetic research to overexpress specific circRNAs, which are a novel class of RNA molecules formed by back-splicing of pre-mRNAs produced by host genes. CircRNAs have been increasingly associated with various biological processes, including tumorigenesis and progression2. In the context of cancer research, vectors like PCD5-ciR are valuable tools for studying the function and regulatory mechanisms of circRNAs
The article discusses experiments conducted to explore the role and mechanism of circular RNA circ_HECTD1 in cell injury after cerebral infarction12. Here’s a breakdown of the key experiments and their purposes:
Expression Analysis: RT-qPCR was used to detect the levels of circ_HECTD1, miR-27a-3p, and FSTL13. This helped establish the relationship between these molecules in the context of cerebral infarction.
Subcellular Localization: A subcellular fractionation assay determined the localization of circ_HECTD1 within cells, suggesting its potential regulatory mechanisms4.
Cell Proliferation and Apoptosis Assays: EdU, MTT, and flow cytometry assays assessed the effects of circ_HECTD1 on cell proliferation and apoptosis, indicating its impact on cell survival after injury.
Protein Level Analysis: Western blot assays measured the protein levels of PCNA, Bcl-2, Bax, PARP, and FSTL1, providing insights into the molecular changes associated with circ_HECTD1 activity5.
Luciferase Reporter Assay: This experiment confirmed the direct interaction between miR-27a-3p and circ_HECTD1 or FSTL1, elucidating the regulatory pathway involved in cell injury.
Each experiment contributed to understanding how circ_HECTD1, through the miR-27a-3p/FSTL1 axis, can affect cell injury mechanisms relevant to cerebral infarction, offering potential therapeutic targets
Subcellular Fractionation & RNase R Treatment: 3
Purpose: To determine the localization and stability of circ_HECTD1.
Result: Circ_HECTD1 was found mainly in the cytoplasm and resistant to RNase R, indicating its circular nature.
Cell Transfection:
Purpose: To knock down circ_HECTD1 and observe the effects on cell proliferation and apoptosis4.
Result: Knockdown of circ_HECTD1 enhanced cell proliferation and reduced apoptosis in OGD/R-treated HT22 cells5.
EdU & MTT Assays:
Purpose: To assess cell proliferative ability6.
Result: Circ_HECTD1 knockdown increased the number of EdU-positive cells and cell viability, indicating improved cell proliferation.
Flow Cytometry:
Purpose: To analyze cell apoptosis.
Result: Circ_HECTD1 knockdown decreased the apoptosis rate in OGD/R-treated HT22 cells5.
Western Blot:
Purpose: To measure the protein levels related to cell proliferation and apoptosis.
Result: Circ_HECTD1 deficiency led to increased levels of PCNA (a proliferation marker) and Bcl-2 (an anti-apoptotic marker), and decreased levels of Bax and Cleaved PARP (pro-apoptotic markers).
Dual-Luciferase Reporter Assay:1
Purpose: To verify the binding relationship between miR-27a-3p and circ_HECTD1 or FSTL17.
Result: Confirmed that circ_HECTD1 directly interacts with miR-27a-3p and that miR-27a-3p targets FSTL1
RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction):
Purpose: To quantify the expression levels of circ_HECTD1, miR-27a-3p, and FSTL1 mRNA in cells affected by cerebral infarction.
Result: The RT-qPCR results would typically show the relative expression levels of these genes. An increase or decrease in the Ct (Cycle threshold) values would indicate changes in gene expression. For instance, a lower Ct value for circ_HECTD1 might suggest higher expression in cells post-infarction, which could be associated with the cellular response to injury
The article describes several experiments to investigate the role of FSTL1 and miR-125a-3p in adipogenic differentiation and inflammation1. Here’s a summary of the experiments and their results:
Isolation and Differentiation of Preadipocytes: Mouse subcutaneous preadipocytes were isolated and induced to differentiate in vitro2. The study observed morphological changes and lipid accumulation, confirming the suitability of these cells for further experiments.
Plasmid Construction and Luciferase Reporter Assay: A plasmid containing the FSTL1 gene was constructed, and a luciferase reporter assay was performed to validate miR-125a-3p targeting the 3′ UTR region of FSTL134. The assay confirmed that miR-125a-3p could bind to the 3′ UTR of FSTL1, reducing luciferase activity.
Transfection of Plasmids, siRNAs, and miRNAs: The study used transfection techniques to overexpress or silence FSTL1 and miR-125a-3p in preadipocytes5. The results showed that overexpression of FSTL1 inhibited adipogenesis and promoted inflammation, while silencing FSTL1 had the opposite effect67.
Oil Red O Staining and Triglyceride Assay: These assays were used to visualize and quantify lipid accumulation in differentiated adipocytes. Overexpression of FSTL1 reduced lipid droplets, whereas miR-125a-3p promoted lipid accumulation8.
Quantitative Real-Time PCR and Western Blotting: These methods were employed to measure the expression levels of adipogenic and inflammatory genes. The results supported the findings that FSTL1 negatively regulates adipogenesis and promotes inflammation, while miR-125a-3p has the opposite effects6.
Overall, the experiments demonstrated that FSTL1 acts as a pro-inflammatory factor inhibiting adipogenesis, and miR-125a-3p can reverse these effects by targeting FSTL1. This suggests potential therapeutic targets for obesity-related inflammatory diseases.
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