Lecture 3 - Transgenic and viral tecnology Flashcards

1
Q

Transgenic Technique Overview

A

Method: Direct injection of DNA into the nucleus of a fertilized egg.
Outcome: Stably incorporates an exogenous gene into cells, making genetic changes heritable.

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2
Q

Applications of Transgenic Animals

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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.

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3
Q

Building a Transgene

A

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.

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4
Q

Promoter Elements and Gene Expression

A

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.

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5
Q

Regulatory Elements (Cis-regulatory)

A

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.

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6
Q

Enhancers and Silencers

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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.

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7
Q

Minimal Promoter

A

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.

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8
Q

Poly(A) Signal (SV40 Processing and Termination Sequence)

A

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.

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9
Q

Poly(A) Tail Functions

A

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.

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10
Q

Poly(A) Signal Sequence

A

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.

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11
Q

Poly(A) Signal Sequence

A

Function: Signals RNA processing machinery to cleave pre-mRNA downstream from the Poly(A) signal.
Polyadenylation: Initiates polyadenylation process.

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12
Q

Methods of Transgene Introduction

A

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.

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13
Q

Introduction into One-Cell Embryo

A

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.

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14
Q

Direct DNA Injection

A

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.

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15
Q

Transgene Expression Challenges

A

Factors: Weak promoter, insufficient regulatory elements, integration at ineffective genomic areas.
Resolution: Assess and modify promoter/regulatory elements for desired expression.

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16
Q

Transgenic Animal Production

A

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.

17
Q

Viral Transduction Using Retroviruses

A

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.

18
Q

Retroviral Genome Components

A

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.

19
Q

Retroviral Infection of Early Embryos

A

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.

20
Q

Use of Viruses in Transgenesis

A

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.

21
Q

Applicability and Limitations of Viral Mediated Transgenesis

A

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.

22
Q

Silencing and Side Effects

A

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.