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.