U01.01.044 Gene expression modifications

Gene expression modifications are vital tools in studying gene function, understanding disease mechanisms, and developing therapeutic strategies. These methods include transgenic strategies in mice, RNA interference, and the use of small RNA molecules to manipulate gene activity.

Transgenic Strategies in Mice

Types of Strategies

Random Insertion of Genes
- Involves integrating the gene at a random site in the mouse genome.
- Results in constitutive expression of the gene.
Targeted Insertion or Deletion of Genes
- Achieved through homologous recombination with a specific mouse gene.
- Allows for conditional expression depending on regulatory elements.

Key Terms

Strategy	Description
Knock-Out	Removing a gene from the genome.
Knock-In	Inserting a gene into the genome.
Cre-lox System	Inducible gene manipulation at specific developmental stages. Useful for studying genes critical for embryonic development.

RNA Interference (RNAi)

RNA interference is a natural cellular process used to regulate gene expression by targeting specific mRNAs.

Mechanisms of RNAi

MicroRNA (miRNA)

Source: Endogenously produced by the cell as hairpin structures.
Function:
- Loose nucleotide pairing allows broader targeting of related mRNAs.
- Blocks translation and accelerates mRNA degradation.
Clinical Relevance:
- Abnormal miRNA expression can contribute to malignancies, such as silencing tumor suppressor genes.

Small Interfering RNA (siRNA)

Source: Usually derived from exogenous double-stranded RNA (e.g., viral RNA).
Function:
- Requires complete nucleotide pairing for high specificity.
- Cleaves mRNA before translation.
Applications:
- Can be synthesized in vitro for gene knockdown experiments.

Comparison: miRNA vs. siRNA

Feature	MicroRNA (miRNA)	Small Interfering RNA (siRNA)
Source	Endogenous	Exogenous
Pairing	Loose pairing	Complete pairing
Specificity	Broader targeting of related mRNAs	Highly specific targeting
Effect	Blocks translation, accelerates degradation	Cleaves mRNA prior to translation
Clinical Role	Linked to malignancies (e.g., tumor suppression)	Used in experimental gene knockdowns

Points to Remember

Random vs. Targeted Insertion:
- Random insertion → constitutive expression.
- Targeted insertion → conditional expression.
Cre-lox System: Enables precise control of gene modification at specific stages.
miRNA: Broad, natural regulation with links to malignancy.
siRNA: Highly specific, experimental tool for gene knockdown.

Bibliography

Lodish et al., Molecular Cell Biology.
Alberts et al., Molecular Biology of the Cell.
Fire, A., & Mello, C. (1998). RNA Interference Studies in Nature.

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