Genetic manipulation 3 Flashcards
What is conventional transgenesis?
direct injection of DNA into the nucleus of a fertilised egg
In a transgenic organisms what cells contain the transgene?
all of the cells
What animals can you make from transgenesis/the uses
express mutant forms of gene
express gene in excess or wrong place
express commercially important products
express genetic markers eg GFP under control of promoters
What 3 things to eukaryotic genes need for building a transgene?
a promoter that will drive expression in the tissues you require
an open reading frame encoding the gene you want to express
sequences that ensure correct mRNA processing
complementary DNA
contains coding region of gene with no introns`
What are “regulatory elements”
DNA sequences known to be bound by transcription factors that activate or repress transcription
4 methods of introducing transgenes into an animal
direct injection with a micropipette
chemical transfection
electroporation
infection
Chemical transfection
Incubate cells in culture medium containing DNA and a chemical that wraps the DNA and is either endocytosed or that diffuses through the cell membrane
What should be done to ensure all cells in the animal in the transgene?
introduce trasngene into 1 cell embryo
Where is the transgene injected into a fertilised egg?
male pronucleus after fertilisation but before nuclear fusion
Nuclear processing
repair mechanisms presumably recognise free DNA in nucleus and try to do something with it - may accidentally ligate transgene
What does long term transgene expression rely on?
transgene integrating with host DNA whichis random
What do transgenes often integrate as?
head to tail concatemer
why may expression not be as expected when the transgenic animals are made?
weak promoter/insufficienct regulatory elements
copy number, position effects, epigenetic modifications, very big trasngene
method for using viruses for getting transgenes into animal cells
retrovirus
reverse transcribed to DNA
integrate into host genome
gag
encodes proteins of nucleoprotein core of virion
pol
encodes reverse transcriptase, integrase etc.
env
encodes surface protein components of virion
what do packaging cells contain?
genes encoding gag, pol and env
what do packaging cells not have?
the packaging signal so the viral cells cannot be included in viruses
are viruses usually used to create transgenic animals in which every cell contains the transgene?
no - usually create a mosaic eg FGF1
viral systems commonly used
adenoviruses
lentiviruses eg HIV
AAV
problems with viral transgenesis
may only work in cells that are dividing
viral sequences subject to silencing mechanisms
side effects - infective new viruses
why are viruses not good at getting DNA integrated into whole animal?
introduced at 1 cell stage often silences
