Transgenic Animals Flashcards
(25 cards)
Name and describe the main technique used to create transgenic animals
nuclear microinjection
directly injecting a small amount of material (transgene) into a fertilised egg.
describe the main stages of nuclear microinjection.
trasngene is injected into the nuclease of the sperm in the fertilised egg.
egg kept in culture during the first few divisions of embryonic development.
embryos implanted into womb of a foster mother
transgene may stably integrate –> Founder animal (or is lost)
why is a male and female founder animal needed?
founder animals contain a SINGLE copy of the trasngene
Male and female founders must be mated to form a new line of animals carrying TWO copies of the transgene (only 25% of offspring).
how is the somatotropin gene from cows used to increase milk production?
somatotropin gene cloned and expressed in E.coli to make recombinant bovine somatotropin (rBST)
–> injected into dairy cows to improve milk production / more milk produced for equivalent feed
how can transgenic livestock be used to produce recombinant proteins? (eg. cows produce protein in the milk)
cloned genes are placed under the control of regulatory regions for ß-casein for expression ONLY in the mammary gland.
product is then secreted in the milk.
relies on casein promotor.
advantages of protein production using transgenic livestock (in milk)
product is only produced in milk, hence less chance of undesired side-effects in host
high expression levels
simple harvesting and purification
name two alternative ways to create transgenic animals (not microinjection)
- retrovirus infection (of fertilised egg)
- embryonic stem cells
briefly explain how retrovirus infection can be used to create transgenic animals
engineered retrovirus vectors infect SOME embryonic cells to introduce transgenes
pros and cons of retrovirus infection of fertilised egg to produce transgenic animals
pros:
- simple
- does not require as much skill as microinjection
- only a single copy of transgene is integrated into the genome
cons:
- viruses can only carry limited DNA
- offspring are always chimeras (not a fully transgenic animal
where are embryonic stem cells derived from? how are they maintained in lab?
the blastocyst
(a very early stage of the embryo, pluripotent so can develop into any body tissue)
must be maintained in conditions that avoid differentiation
how are embryonic stem cells used to generate new transgenic animals?
engineered ESC’s are inserted into the central cavity of an early embryo at blastocyst stage.
results in the creation of a chimera consisting of some transgenic and some normal tissue.
why do transgenic animals carrying the same inserted transgene often differ in levels/patterns of expression?
the LOCATION of the transgene (where it is inserted) is different!
(eg. poorly expressed if inserted in heterochromatin, which is a gene-poor transcriptionally-inactive form of chromatin)
transcription is also affected by nearby regulatory elements
how can we combat location effects on transgene expression? name two strategies
- incorporate appropriate REGULATORY ELEMENTS into the construct
- target insertion to specific site
two types of regulatory elements to incorporate into transgene constructs to combat location effects
- enhancer sequences - dominant up-regulation, drives downstream expression.
- Insulator sequences (boundary elements on both ends of transgene, blocks signals from other regulatory sequences near site of insertion)
give an example of an enhancer sequence for the ß-globin gene cluster.
locus control region (LCR) - hypersensitive sites upstream of the structural genes of the ß-globin cluster
confers HIGH EXPRESSION
note: distinct from individual promotors.
what is the method by which transgene insertion can be targeted to a specific site? DRAW A DIAGRAM TO EXPLAIN THIS.
Targeting vectors - operate by homologous recombination
(from previous bio: two chromosomes, one paternal, one maternal. cross and some sections recombine. similar method)
vector has sequences that are homologous to the insertion site of host chromosome. vector linearises, ends cross and inserts itself.
can also be used to replace.
reasons why controlled transgene expression is useful
for protein production, high expression is desired, however:
some products are toxic in high amounts
gene expression should also be kept low when establishing a line of transgenic animals.
controlled expression is useful for functional analysis.
how is transgene expression controlled?
by choosing the promotor
name three types of promoters used to control transgene expression
inducible endogenous promoters
recombinant promotor systems
steroid receptor systems
what are inducible endogenous promotes?
natural promoters in host’s DNA that can be switched on and off in response to chemical / environmental stimuli
eg. metallothionein induced by heavy metals
heat shock promoters (active above a certain T)
what are recombinant promoter systems used for controlled expression of transgenes?
modified bacterial promoters
eg. LacI, TetR repressors
transcription is blocked when repressor is bound
Describe steroid receptor systems (for controlling transgene expression). what are the advantages of such a system?
steroids are lipophilic hence penetrate cell membranes rapidly
steroids bind to receptor proteins, which then bind directly to DNA and regulate gene expression.
short half life of a few hours
how can we avoid inducing other host genes when using a steroid controlled transgene?
choose a steroid that is not naturally found in the host animal
eg. ecdysone from insects
After the transgene has been incorporated in the host animal, another way to control transgene expression is site specific recombination, removing or reversing DNA segments to achieve activation of transgene. how is this done?
by recombinase systems
Eg. Cre/loxP as seen previously to remove antibiotic resistance selectable markers
can also be used to remove blocking sequences, for conditional knockouts etc
requires one animal with DNA flanked by loxP, to mate with one animal with the Cre gene driven by a specific promotor.
