Genetics Notes

Gene Knockout and Targeting

Background Matters: The same mutation can manifest differently based on the genetic background (e.g., white mice vs. black six mice).
- Example: A gene knockout results in a fat mouse in one strain but a mouse unable to walk in another.
OB Gene and Leptin: Knocking out a gene (like the OB gene) helps determine its function.
- The OB gene produces leptin, a hormone regulating body weight and appetite.
- Mice without the OB gene don't produce leptin, leading to a persistent state of hunger and morbid obesity.
Gene Targeting and Knockout Animals: This involves breaking a gene and observing the resulting phenotype to understand the gene's normal function.
Human Relevance: Morbidly obese people without psychiatric disorders often have mutations in the leptin gene.
- Leptin can be administered to mice to control hunger.
Gene Targeting: Targeting a specific allele locus or promoter to learn about a gene of interest (e.g., the OB gene).
Gene Knockout: Generating a null allele or loss of function.
Phenotype Observation: Observing the phenotype (e.g., mice with short limbs) reveals the normal function of the gene (regulating limb length).

Targeting Vector: A construct nearly identical to the target gene (or exon) but with a neomycin resistance marker inserted to disrupt its function.
Process: Introduce the targeting vector into embryonic stem cells (ES cells).
Recombination: At low frequency, homologous recombination occurs, inserting the disrupted gene into the genome.
Selection: Select for successful recombination events using neomycin resistance.
- ES cells are grown in the presence of G418; only cells with the neomycin resistance marker survive.
Blastocyst Injection: Inject the selected ES cells into a mouse blastocyst.
Surrogate Mothers: Implant the blastocyst into pseudopregnant females.

Targeting Vector with LoxP Sites: Contains a gene with LoxP sites flanking the neomycin resistance marker.
Cre-Lox Recombination: LoxP sites allow removal of the neomycin marker using Cre recombinase, creating a clean knockout.
Null Allele Creation: Removal of a critical exon results in a null allele.
Knock-ins: Involve adding something to the gene (e.g., an exon or GFP).
Thymidine Kinase (TK): Used to force recombination by selecting against random insertion.
- Cells with TK die in the presence of gancyclovir, ensuring only cells with targeted recombination survive.

Purpose: Confirm that the gene targeting occurred correctly.
Method: Use Southern blots with different probes and restriction enzymes to verify the targeted locus.

Chimeric Mice: Generated by injecting ES cells from one color mouse into a blastocyst from another color mouse.
- Striped progeny indicate successful contribution from the ES cells.
Homozygosity: Cross chimeras to achieve homozygosity for the knockout allele.

Promoter Expression: Fuse a promoter to GFP to create a transgenic mouse and observe where the promoter is expressed.

HPV and Gender Neutrality: HPV vaccine is recommended for both genders.
- Protects against cervical, esophageal cancers, and genital warts.
- Protects against other types of cancer beyond cervical cancer.
Viral Contribution: Approximately 12% of human cancers are associated with viruses.
Multifactorial Nature: Virus infection alone is insufficient; DNA damage and accumulation of mutations are also required.

Genome Instability: Cancer cells exhibit inherent instability due to abnormal cell division.
Lagging Anaphase Chromosomes: Chromosomes lag during anaphase in some cancers.
- Specific cancers show recurring gains and losses of chromosomes.
Progressive Nature: Multiple mutations are generally required for cancer development.
- Colonoscopies are recommended to remove polyps before they become cancerous.
Retinoblastoma: An exception to the progressive nature, explained by the two-hit theory.
- Individuals born heterozygous for a tumor suppressor gene have an increased risk of developing cancer.

Tumor Suppressors: Suppress tumor formation; if broken, tumors develop.
Proto-oncogenes: Regulate cell cycle, differentiation, division, and apoptosis.
- Promote cell division but must be regulated.
Cell Division Regulation: Cells must divide to heal wounds but also stop dividing when the wound is healed.
Mutations: Mutations in proto-oncogenes turn them into oncogenes.

Radiation: Uses a focused beam to cause DNA damage and induce apoptosis in cancer cells.
Chemotherapy: Affects all cells, causing severe side effects; not a targeted therapy.
Targeted Therapy (Herceptin): Only effective for HER2-positive breast cancers.
- Binds to HER2 receptors and induces cell death.
- Expensive and requires early detection.
Antibody Therapy: Herceptin (Trastuzumab) is a monoclonal antibody that targets the HER2 receptor.
Precision Medicine: Tailoring treatment based on the multifactorial nature of cancer and individual patient characteristics.
- Involves sequencing the genome, analyzing mutations, and assessing proteomics.

Philadelphia Chromosome: A translocation resulting in a BCR-ABL fusion, correlated to a specific type of cancer.
Chronic Myelogenous Leukemia (CML): Managed but not cured; can modulate with compounds like Gleevec (imatinib).
- Gleevec blocks the ATP binding site, reducing cell division activity.
Gleevec Mechanism: Imatinib binds to the ATP binding site of the BCR-ABL fusion protein, inhibiting its activity and reducing cell division.
Monitoring and Management: Patients monitored for life with potential switches to alternate TKIs if resistance develops.
Bone Marrow Transplant: Allogenic stem cell transplantation is a last resort.

Gene Amplification: Looking for gene amplification indicates targeting locus has been reached.