Huang Lectures Flashcards
Gene Manipulation with Mutagenesis
- the process begins with P0 generation exposed to a mutagen, creating genetic variations.
- these modifications are passed down to F1 and F2 generations.
- common wild-type phenotype and rare mutant phenotype differences allow researches to identify genes responsible for specific traits.
What is ‘functional genomics’? and what other fields does it integrate?
- functional genomics uses genome sequence data to understand gene functions and how they interact within biological systems.
- other fields integrated: genomics, molecular biology, bioinformatics.
Forward Genetics
- starts with natural variation or induced mutagenesis
- moves from phenotype (observable trait) to genotype (underlying genetic cause) to pinpoint genes associated with specific phenotypes.
Reverse Genetics
- involves targeted gene modification
- starts with known genotype and investigates the resulting phenotype to understand the gene’s function.
Gain of Function Genetic Screens
- activation/enhancement of gene function
- DNA transfection
- cDNA/ORF
- Insertional mutagenesis
- CRISPRa
- gain of function screens aim to identify genes that, when over expressed or mutated lead to specific phenotypes such as uncontrolled cell growth.
Loss of Function Genetic Screens
- inactivation/suppression of gene function
- classical gene knockout
- insertional mutagenesis
- RNAi
- CRISPR knockout
- CRISPRi
- Cas13d, Cas 7-11 - mediated gene supression
Altered function - genetic screens
- other CRISPR editing tools - DSB mediated, base editing, prime editing.
Example of DNA Transfection (GoF Genetic Screen)
- Human HRAS oncogene
- normally, Ras protein switches between inactive (Ras-GDP) and active (Ras-GTP) states, regulating cell growth and migration.
- mutation-induced gain of function: an increase in the active form (Ras-GTP) can disrupt balance, leading to uncontrolled proliferation and cancer.
Pros & Cons of
cDNA over-expression (GoF Genetic Screen)
1) Pros:
- effective, adaptable, and enables systematic over-expression.
2) Cons:
- can produce experimental artifacts like cloning errors (e.g., gene truncations or mutations) and can cause abnormal expression levels due to the overuse of strong promoters or genes not typically expressed in certain cell types.
What are two types of Insertional Mutagenesis?
- Virus-Mediated:
- Viral vectors (e.g., lentiviruses) insert genes with a strong promoter (viral LTR) into the host genome, causing random mutations.
- These insertions may activate or inactivate genes by adding enhancers, promoters, or disrupting gene coding regions.
- Transposon-Mediated:
- DNA transposons like PiggyBac and Sleeping Beauty can jump into new DNA locations, driven by transposase enzymes.
- This approach enables reversible integration, where transposable elements can deliver genetic elements like promoters, splicing sequences, or transcriptional stops
What is Lentiviral Transduction?
Lentiviral vectors are often used to deliver and integrate genetic material:
- Creating the Lentivirus: A laboratory procedure to package the gene of interest into a viral vector.
- Infecting Target Cells: The virus is used to infect cells, ensuring the gene of interest integrates into the target cell genome.
Cancer Gene Discovery by Insertional Mutagenesis
- This approach uses insertional mutagenesis to uncover potential cancer-causing genes.
1) Screening Process:
- Insertion Methods: Retroviral infection, transposon mobilization, or lentiviral transduction introduce genetic elements randomly into the genome.
- Tumorigenesis: Mutagenized cells are observed for tumor formation.
- Gene Identification:
- After tumor development, DNA extraction is performed.
- Vector-genome junction amplification allows the identification of insertion sites.
- Sequencing and mapping reveal potential candidate cancer genes.
Validation Process!!
* Transformation Assays: Both in vitro and in vivo assays assess the cancer-causing potential of identified genes.
- Oncogenomic Cross-Species Analysis: Helps to validate findings and identify therapeutic targets and biomarkers.
-Pros:
* Straightforward setup with simple machinery.
* Suitable for in vivo screens and allows incorporation of larger DNA segments.
- Cons:
* Integration-site dependency: Where the vector inserts in the genome can influence results and limit predictability.
What are pros & cons of CRISPR activation systems?
1) Pros:
- Sequence-specific and reversible: CRISPRa can activate native genes without altering DNA sequence permanently.
- Equal representation: Can target genes uniformly across a population of cells.
2) Cons:
- Guide RNA dependency: Requires well-designed sgRNAs for effectiveness.
- Challenges with chromatin: Densely packed chromatin regions can block access to target sites.
- TSS Annotation Requirement: Needs accurate transcription start site (TSS) information for precise activation.
What are pros & cons of gain of function screens?
1) Pros:
- Stable, versatile, and creates dominant phenotypes, making gene function more observable.
2) Cons:
- Non-physiological expression artifacts: Overexpression may not reflect natural gene behavior.
- Size limitations: Large DNA sequences are challenging to introduce using lentiviral packaging, particularly for full-length cDNAs.
Loss of function screens: Gene Knockout (KO) in simple eukaryotes
1) Engineered DNA in Yeast: Uses KO techniques to test gene essentiality.
- Yeast cells can be either haploid, making complete knockouts simpler, or diploid, allowing flexibility in genetic studies.
2) Challenges in Mammalian Cells: Before RNAi and CRISPR, gene knockouts were slow and low-throughput, not practical for systematic or high-throughput studies.
