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).

Creating Knockout Animals

• 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.

Detailed Knockout Process

• 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.

Southern Blots for Confirmation

• Purpose: Confirm that the gene targeting occurred correctly.

• Method: Use Southern blots with different probes and restriction enzymes to verify the targeted locus.

Chimeras and Homozygosity

• 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.

Practical Applications

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

Viral Association with Cancer

• 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.

Hallmarks of Cancer

• 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.

Proto-oncogenes and Tumor Suppressors

• 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.

Cancer Therapies

• 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.

Specific Cancer Examples

• 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.

Visual Confirmation

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