gentics lecture 9

The primary focus of the discussion is on reverse genetics, particularly through mouse models.
Emphasis on the anticipation of not revisiting previous screening methods extensively.

Mice as the gold standard for translational genetic models in genetics relevant to humans.
The expectation to develop mutant mice for studying protein effects in higher mammals.
The mouse model facilitates investigations into genetic underpinnings of various processes due to the similarity of mouse and human genetics.

Short gestational cycle: approximately 3 weeks.
Maturity reached in 6 to 8 weeks, leading to the need for several months to evaluate mutations and potential reproduction.
Structural and genetic similarity to humans is significant, surpassing many other organisms.

Mention of the Repository for Mutant Mice storing around 60,000 different strains cryogenically.
Process includes harvesting embryos, cryofreezing, and re-implanting them into foster mothers for breeding.
Users of the repository must pay a fee (e.g., approx. $4,000 for a cryogenic recovery of mutant mice), which is not covered under non-profit obligations.

Labs either create mutant mice in-house or utilize repositories based on their resources.
Making mutant mice in-house is a resource-intensive process that might take a significant amount of time (potentially up to or exceeding a couple of years).

Fundamental breakthroughs in creating mutant mice stem from:
- Homologous recombination to modify DNA accurately.
- Harvesting embryonic stem cells from early-stage embryos allows modification and lateral introduction into embryos to create genetically modified mice.

Description involves creating a targeting vector with specific homeologous arms flanking the modification site.
Targeting vectors may include selection markers (e.g., antibiotic resistance genes) to isolate successful modifications efficiently.
A description of the neomycin resistance cassette, and thymidine kinase (TK) system is provided:
- The neomycin resistance cassette provides a selection mechanism (cells with successful gene integration survive neomycin).
- TK makes cells sensitive to gancyclovir, allowing for the elimination of cells without successful homology.

The simultaneously positive and negative selections ensure the efficiency of homologous recombination (target gene correctly modified).
Failures (illegitimate recombination leading to TK cassette integration) lead to cell death, ensuring accuracy in target isolation.

Describes the complete process from isolating stem cells from a developing mouse to implantation:
  - Isolation from the inner cell mass of an embryo.
  - Modification of the genetic content through targeted means.
  - Injection into embryos with a specific color gene for tracking (e.g., agouti coat color for identification in offspring).

Modified embryonic stem cells will produce a chimeric mouse, with mixed genetic outcomes (e.g., patches of different coat color).
Breeding strategies used to identify successful germ-line transmission of genetic modifications.

Outcomes may include all black offspring (no incorporation), agouti patches (chimeric incorporation), or diverse originally black results which indicate wild type (no modification).
Analysis through genotyping methods (e.g., PCR) to confirm genetic status then allows tracking of inheritance patterns.

Discussion on CRISPR and RNA interference as gene expression manipulation techniques.

Importance of CRISPR/Cas9 as a revolutionary gene editing tool.
- The CRISPR system allows for efficient and flexible genetic modifications in organisms, driven by a synthetic guide RNA.
Origin: 2011 discovery led by Jennifer Doudna and Emmanuelle Charpentier.
- The tool's utilization emerged quickly across research due to its ease of use and cost-effectiveness, potentially reducing the timescale in producing genetically modified organisms significantly compared to previous methods.

Cas9-derived protein's function hinges on targeting DNA based on guide RNA coding sequences.
Multiple CRISPR-associated proteins are involved in processing the RNA, recognizing, and cleaving target DNA effectively.

RNA interference (RNAi) allows for precise gene expression modulation.
- Key players include Dicer and RISC/Argonaute, facilitating mRNA degradation or translational repression.
- Deploying RNAi can manifest as gene knockdown effects for experimental or therapeutic purposes.

CRISPR as an avenue to explore potential treatments for genetic deficiencies like sickle cell disease and beta thalassemia through genetic modification of hematopoietic stem cells.
Advances in CAR-T cell therapy illustrate CRISPR's application in cancer treatment.

Utilization raises important ethical questions around human gene editing, particularly in relation to inheritable changes and consent.
Current popularity has prompted discussions on revising gene editing laws and regulations to improve oversight and safe application for potential human therapeutic contexts.

Overview reflects the dramatic evolution of genetic manipulation techniques from homologous recombination to RNA interference and CRISPR, showcasing their efficiency, application, and evolving integration into modern genetic research. Moreover, the implications of these advancements and their ethical considerations underscore the necessity for continual dialogue within the scientific community to refine methodologies while ensuring safety and integrity in research applications.