Transgenic 1 Flashcards
What is a Transgene?
- Gene/genetic material transferred from one organism to another (naturally or using genetic engineering) – generally germ line
A TRANSGENE May change the phenotype of the
organism its introduced into.
New gene may: 2
– retain the ability to produce RNA/protein
– alter the normal function of the t/g organism’s genetic code
WHY MAKE A TRANSGENIC ANIMAL? 5
1 *Identification of gene function
2 *Generation of animal models of diseases
3 *Drug validation
4 *Cell and organ research
- Others
Improvement of livestock WAS limited by?
NOW WHAT HAPPENS?
Improvement of livestock -traditionally limited by NATURAL VARIATION…
NOW- ‘make’ animals - express a trait of interest …
What are the 3 basic techniques to produce TRANSGENIC MICE:
(1) Pronuclei injection
(2) Retroviral gene transfer
(3) Embryonic stem cells - gene target
Transgenesis - history …
- jaenisch + Mintz,1976
infected early mouse embryos; Moloney retrovirus -
- mice integrated viral DNA into all tissues + their germlines
(passed on to offspring) .. first transgenic mammals. - Gordon and Ruddle, 1980
mouse born with genetic material injected into
pronuclei of a fertilised mouse egg - The term “transgenics” was born …
TRANSGENE IS THE INTRODUCED DNA…
why is mouse USED AS THE MODEL? =7
1 * Embryos are readily obtained and easy to
maintain.
2 * Developmental period is relatively short.
3 * Many offspring.
4 * Relatively inexpensive to maintain.
5 * Genetics are well established and genome
has been completely sequenced.
6 * Embryonic stem cells are available.
7 * Useful for human research – tissues &
organs similar, carry most of the same genes
What can FOREIGN DNA be used for in TRANSGENESIS? 5
1 * over-express protein (study the effects of
increased levels of a gene)
2 * express a defective protein
3 * express a gene product that adversely affects
the WT gene product (dominant negative)
4 * inactivate an endogenous gene (KO)
- conditionally inactivate (conditional KO)
- tissue-specific KO (Cre-Lox)
5 * replace endogenous gene with another gene
(KI)
What is gene locus?
Gene Locus: Includes both the promoter and the transcription unit!
Parts of a Transgene - Recombinant DNA
PROMOTER
1. Distal (>100kb)
- Proximal (approx. 1kb)
TRANSCRIPTION UNIT:
- Coding and noncoding sequences (from 1kb to >200kb)
Promorter/enhancer sequences drive expression?
4
Promoter/enhancer sequences
….drive expression …
- tissue
- time
- amount
- control via regulatory elements
How are TRANSGENES DEVELOPED AND INCORPORATED?
1 * Transgenes are usually developed **from plasmids, and include a promoter region (which may be tissuespecific) and a coding sequence, usually a **cDNA for the gene of interest.
2 * The plasmid is LINEARISED and INJECTED into the PRONUCLEUS of a fertilized OOCYTE .
This is then implanted
into PSEUDOPREGANANT FEMALES
3 * Transgenes are incorporated RANDOMLY into the genome, and are INFLUENCED BY THE POSITION at which they integrate
Transgenic Technology
- PROMOTER VS cDNA examples
PROMOTER
1- Tissue specific (brain, liver, muscle …)
2- Ubiquitous
3- Inducible (tetracyclin, interferon…)
cDNA
- GAIN OF FUNCTION:
- wild-type gene
- mutant
- LOSS OF FUNCTION:
- dominant negative
- antisense
What is the USE OF REPORTER GENES? HOW (4)
EXAMPLES OF VISUALLY IDENTIFIABLE ONES: 3
THEY Indicate successful uptake of the transgene:
1 - Distinct from endogenous gene
2 - Level of expression
3 - Ease of detection
4 - Study localisation
VISUALLY IDENTIFABLE:
- B galactosidase
- Luciferase
- GFP
- others…
Genetically altered Mice:
KNOCKOUTS…
What is the in vivo function of a gene? -
a) What is the in vivo function of a gene? -
knocking out the activity of a gene provides information about what that
gene normally does
b) Develop a model of disease (only where the disease is a result of loss of function) – develop & test novel therapies (cancer, obesity, CHD, diabetes, anxiety,M Parkinson’s)
Pronuclei Injection
WHAT IS IT? PURPOSE? 3
- Introduce foreign DNA (the transgene) into the pronucleus (nucleus of a sperm/egg cell during fertilization)
- Get random incorporation of the DNA = ECTOPIC INSERTION
- Hopefully ~10-40% of injected pronuclei contain construct transgene will be expressed in the germline & generate a strain of animals expressing the desired gene
Pronuclei Injection DNA is… 3
DNA
1. very clean
- no vector sequences
- promoter - reporter
Pronuclei Injection METHOD?
How many mice and why do need them?
Need 4 sets of mice:
(1) donor females - collect fertilised eggs
(2) stud males - produce fertilised eggs
(3) foster females - pseudopregnant recipients
(4) sterile males –vasectomised (mate with foster mothers)
- Need to induce egg formation and release in donor females (superovulated) - hormone injection.
- Plugging occurs, recover 20-30 oocytes/mouse & inject with genetic material
-Implant into pseudopregnant female
GENERATING A TRANSGENIC MOUSE: How does it work..is it due to amount of DNA or expression
- The SAME AMOUNTOF GENE (dosage) is present in all cells.
- Its LEVEL OF EXPRESSION is
CONTROLLED BY PROMOTER. - However, its EXPRESSION
may be INFLUENCED by its
POSITION IN GENOME.
(positional effect)
STEPS IN GENERATING A TRANSGENIC MOUSE? 5
- inject foreign DNA into one of the pronuclei.
- Fertilised mouse egg prior to fusion of male and female pronuclei.
- Transfer injected eggs into foster mother
- About 10 to 30% of offspring contains injected foreign DNA. Foreign DNA is present in equal amounts in all tissues.
- Mice expressing foreign DNA are BRED to continue the DNA in germ line.
How is transgenic mouse identified?
- labs
Transgenic mouse EMBRYO in which the PROMOTER for a GENE EXPRRESSED in NEURONAL PROGENITORS (neurogenin 1) DRIVES EXPRESSION OF A BETA-GALACTOSIDASE REPORTER GENE.
NEURAL STRUCTURES expressing the REPORTER transgene are DARK BLUE-GREEN.
Disadvantages of pronuclei injection: 3
1 * can’t control where/ # copies transgene integrate in
mouse genome = random insertion/multicopy arrays
- promoter/enhancer effects
- position effects; disruption of other genes
2 * presence of the endogenous gene
3 * can’t select ‘positives’ - 3 weeks postpartum
Embryonic stem cells overcome the problems of…
Pronuclei Injection
What are EMBRYONIC CELLS?
1981 - isolated ES cells
1 * stem cells from blastocyst (early embryo)
2 * can maintain an undifferentiated state (renew)
3 * phenotype of early embryonic cells (pluripotent - differentiate into any embryonic cell type)
- can participate in generation of germ-line chimeras when introduced into a mouse blastocyst
Chimera = organism composed of tissues containing two or more genetically distinct cell types
- genes can be introduced into ES cells via transfection,
retroviral infection, injection, electroporation
