Lecture 3 - Transgenic and viral tecnology Flashcards
Transgenic Technique Overview
Method: Direct injection of DNA into the nucleus of a fertilized egg.
Outcome: Stably incorporates an exogenous gene into cells, making genetic changes heritable.
Applications of Transgenic Animals
Purpose: Express mutant forms of a gene, overexpress a gene, produce commercially important products (e.g., insulin in milk), express genetic markers like GFP under interesting promoters.
Flexibility: Allows experimentation with target genes.
Building a Transgene
Components: Promoter, open reading frame (ORF) encoding the gene to express, sequences ensuring correct mRNA processing.
cDNA: Isolated from the gene, contains only coding regions (no introns).
Promoter Elements: Incorporated to drive gene expression in specific tissues.
Promoter Elements and Gene Expression
Promoter Elements: DNA sequences upstream of a gene’s coding region.
Function: Binding sites for transcription factors and RNA polymerase, initiating transcription.
Core Promoter: Minimal region with essential elements (e.g., TATA box) where RNA polymerase binds.
Regulatory Elements (Cis-regulatory)
Definition: DNA sequences controlling gene expression by interacting with transcription factors and regulatory proteins.
Location: Can be near or far from the gene.
Function: Act as enhancers or silencers, influencing gene activation and repression.
Enhancers and Silencers
Enhancers: Bind to transcription factors, enhancing gene expression; can be located anywhere in the gene.
Silencers: Bind to specific proteins, repressing gene expression and inhibiting transcription.
Minimal Promoter
Definition: Smallest DNA sequence initiating transcription at a basal level.
Composition: Includes core promoter element, lacks regulatory elements/enhancers.
Function: Represents minimal requirements for transcription initiation.
Poly(A) Signal (SV40 Processing and Termination Sequence)
Essential Element: Found in eukaryotic DNA, named after Simian Virus 40.
Role: Crucial in post-transcriptional processing and termination of mRNA.
Termination Signal: Recognized by RNA polymerase II, triggers mRNA maturation.
Polyadenation: Adds a long chain of adenine nucleotides to the 3’ end of pre-mRNA during transcription.
Poly(A) Tail Functions
Stabilization: Protects mRNA from degradation by exonucleases.
Export: Aids in mRNA export from the nucleus to the cytoplasm.
Translation Initiation: Interacts with initiation factors during translation initiation.
Poly(A) Signal Sequence
Stabilization: Protects mRNA from degradation by exonucleases.
Export: Aids in mRNA export from the nucleus to the cytoplasm.
Translation Initiation: Interacts with initiation factors during translation initiation.
Poly(A) Signal Sequence
Function: Signals RNA processing machinery to cleave pre-mRNA downstream from the Poly(A) signal.
Polyadenylation: Initiates polyadenylation process.
Methods of Transgene Introduction
Direct Injection: Transgenes injected into DNA of cells.
Chemical Transfection: Cells incubated with DNA and a wrapping chemical in culture medium.
Electroporation: Introduction of DNA using electric fields.
Virus Exposure: Cells exposed to viruses carrying transgene DNA.
Introduction into One-Cell Embryo
Objective: Ensure transgene in all cells of an organism.
Approach: Introduce transgene into one-cell embryo (fertilized zygote).
Outcome: Mosaic organism if introduced into an 8-cell embryo.
Direct DNA Injection
Timing: Injection into male pronucleus after fertilization but before nuclear fusion.
Repair Mechanisms: Nucleus repair mechanisms may recognize and integrate transgene DNA.
Integration: Usually random integration with the host DNA.
Transgene Expression Challenges
Factors: Weak promoter, insufficient regulatory elements, integration at ineffective genomic areas.
Resolution: Assess and modify promoter/regulatory elements for desired expression.
Transgenic Animal Production
Procedure: 20 transfers of transgene oocyte into pseudopregnant mothers result in 4-5 pregnant mice, yielding 40-50 embryos.
Transgene Carriage: 4-15 embryos may carry the transgene.
Expression: 0-15 embryos may express the transgene.
Viral Transduction Using Retroviruses
Process: Retroviruses infect cells, and RNA is reverse-transcribed into DNA for integration.
Virus Components: Gag, pol, env, and Ψ elements in a typical retroviral genome.
Function: Delivers transgenes into infected cells for integration into the host genome.
Retroviral Genome Components
Gag: Encodes proteins of the nucleoprotein core of the virion.
Pol: Encodes reverse transcriptase, integrase, and their functions.
Env: Encodes surface protein components of the virion.
Ψ Element: Packaging signal in the retroviral genome.
Retroviral Infection of Early Embryos
Process: Plasmid engineered to express transgene flanked by retroviral sequences.
Transfection: Introduced into a cell line expressing gag, pol, and env.
Viral Particle Formation: Transgene packaged into infective particle with reverse transcriptase and integrase.
Exposure: Embryos or cells exposed to infective viruses containing the transgene.
Use of Viruses in Transgenesis
Mosaic Creation: Viruses commonly used to create mosaics in mid-gestation embryos.
Adenoviruses: Large DNA viruses suitable for transient transfection but eliminated by the immune system.
Adeno-Associated Viruses (AAV): Smaller, non-pathogenic, can infect non-dividing cells, popular for gene therapy.
Lentiviruses: Derived from HIV, efficient at infecting dividing and non-dividing cells, stable integration, suitable for long-term expression.
Applicability and Limitations of Viral Mediated Transgenesis
Applicability: All organisms, popular for non-mainstream animals like chickens.
Limitations: May only work in dividing cells, subject to host defenses (silencing), potential for side effects (recombination leading to new viruses).
Integration Issues: Viruses are efficient in specific tissues but not good at integrating DNA into the entire animal.
Silencing and Side Effects
Silencing Mechanisms: Viral sequences introduced at the 1-cell stage often silenced.
Direct DNA Injection: Efficient but may occur at uncontrolled integration sites, posing a risk of unwanted mutations.
