BIO2015: Lecture 5

  • Created by: LMoney
  • Created on: 12-05-14 13:10
what is germ-line modification?
affects every cell, inherited by next generation
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germ cells and what kind of cells separate early in animal development?
germ cells
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To achieve germ-line transformation, when does DNA need to be introduced?
before germ cells form- usually egg or early embryo
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Describe microinjection as a DNA transfer method
usually used on single cell. Effective but inefficient
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describe particle bombardment as a DNA transfer method
a.k.a gene gun, used more in plants because they have tougher cells, use DNA-coated cell
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describe DNA/calcium phosphate precipation and other chemical methods
cheap but inefficient
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describe Phospholipid vesicles - liposomes (lipofection)
Efficient, many commercial products use this process of DNA transfer
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describe electroporation as DNA transfer method
reliable, efficient, increasingly refined (specialised machine required)
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describe viral carriers as DNA transfer method
increasingly common, very efficient. Introduce DNA into cells by transduction
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what are the 2 main methods for creating transgenic mice?
1) DNA injection into nucleus of fertilised egg - pronuclear injection 2) Homologous recombination in embryonic stem cells - ‘gene targeting’
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what does the method of pronuclear injection use?
newly fertilised mouse egg, male and female pronuclei visible- normally inject into larger male pronucleus
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which gene was fused to rat or human growth hormone gene in making gigantic mice?
metallothionein-I gene promoter (metal metabolism)
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what were the mice fed to induce the transcription of this gene?
zinc- transgenic were larger and heavier
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what did transcription of the transgene cause?
caused high blood concentrations of growth hormone protein in transgenics
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what are the problems with pronuclear injection?
1) Can only be used to add genes, not to modify existing genes 2) Insertion in random locations (ectopic) and multiple copies 3) ‘Position effects’ of flanking chromosomal sequences – sometimes get ‘silencing’ or enhancement
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what is the implication of these 'position effects'?
expression of transgene will vary depending on its position in the genome
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when was homologous recombination in embryonic stem cells developed?
late 1980's
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what did this method allow?
precise modification of specific genes
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how does this method work?
A single copy of the modified gene is modified in its normal chromosomal environment
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which natural process does this method rely on?
homologous recombination- also occurs as a natural repair mechanism in non-dividing cells- it involves exchange of a DNA segment between DNA strands
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when was the method of embryonic stem cells for DNA transfer developed?
early 1980's
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what do embryonic stem cells from the blastocyst inner cell mass?
develop into embryo
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are they easy to isolate in mice compared to other species?
yes, easier to isolate in mice
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what is special about stem cells?
capable of developing into any cell type (including germ cells)
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what do stem cells have a higher rate of?
homologous recombination
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How can we use stem cells to make a transgenic mouse?
gene targeting
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what does gene targeting use?
Linearised plasmid cloning vector (cut once with a restriction enzyme)
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describe the process of gene targeting?
5’- and 3’-arms contain homologous (identical) sequence to the gene to be targeted in the chromosome - targeting vector will line up with the chromosome and homologous recombination will replace the region in-between the 5’ and 3’ arms
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why are markers included?
to allow selection of stem cells that have incorporated transgene successfully
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what is the Neo gene for?
neomycin resistance marker will be incorporated if successful- neomycin is an antibiotic so resistance against it will aid selection
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which gene marker will be left behind if homologous recombination is successful?
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how does positive and negative selection improve efficiency?
by killing ES cells that have not incorporated transgene, or not in correct position
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which exon does neoR disrupt?
Exon 2- preventing translation of the protein- the gene will be knocked out
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Presence of neoR(neomycin resistance gene) is tested by what?
G418, a neomycin analogue that normally kills the cell. When neoR is present cell is resistant to G418- positive selection
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what is presence of tk (thymidine kinase gene) is tested by?
gancyclovir, a toxic thymidine analogue. When tk is present gancyclovir is incorporated into DNA, and the cell dies (negative selection)
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Homologous recombination replaces (blank) gene with mutated sequences?
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(blank) random insertion of the vector does not replace endogenous gene
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germ cells and what kind of cells separate early in animal development?


germ cells

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To achieve germ-line transformation, when does DNA need to be introduced?


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Card 4


Describe microinjection as a DNA transfer method


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Card 5


describe particle bombardment as a DNA transfer method


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