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
CHIMERA?
Chimera = organism composed of tissues containing two or more genetically distinct cell types
Generating a KO mouse
- producing the ES Cell
- MORULA:
8 cells = 2 1/2 days - MORULA
16 cells = 3 days - SELECTION OF BLASTOCYST (inner cel mass, blastocoel, Trophectoderm)
- in a CONTAINER = EMBRYONIC SREM CELLS + IRRADIATED FEEDER CELLS
THE ADAVANTAGES AND JOBS OF EMBRPNIC CELLS?
- Can direct transgene to endogenous gene = gene targeting/homologous recombination
- can check recombinants before ‘making’ mice
EMBRYONIC CELLS HAVE Two distinct advantages over embryos …
- SINGLE copy of transgene inserted
- identify clones expressing transgene prior to
producing animals
Targeting Constructs for Knockouts PURPOSE OF NEO
Neo cassette allows
positive selection for cells that incorporate the targeting construct.
Neo cassette
confers resistance to neomycin analogs, such as G418. Cells with no Neo will die in the presence of G418.
Targeting Constructs for Knockouts PURPOSE OF THYMIDINE KINASE (TK)
Thymidine kinase (TK)
cassette allows negative
selection - cells that have this present have not undergone homologous recombination.
TK cassette confers
sensitivity to gancyclovir,
which will kill the cells
ES CELLS - GENE KNOCK OUT:
PRODUCTION OF ES CELLS WITH A GENE KNOCKOUT
- Closed gene: with Exon 1 and 2
- ADD tk+ GENE
INSERT neo^R into EXON 2 - Targeting vector
tk+
NEO^R - ADDING TARGETING VECTOR
- CULTURED MOUSE EMBRYONIC STEM CELLS
ES Cells - Targeted insertion
Homologous Recombination:
POSSIBLE OUTCOMES
- Vector
Target gene in chromosome
–> neo^R tk-
Chromosome with targeted insertion - Ectopic random insertion
Vector
—>non-target gene in chromosome
tk+ neo^R
Chromosome with random insertion - no insertion
vector
nontargt gene in chromsome
–>
Unchanged chromosome
tk- neo^s
Embryonic cell SELECTION
- Selection of cells with gene knockout
- Neomycin analog = kills neo^S cells + Ganciclovir = kills tk+ cells
- —-> Add to MEDIUM
- in MEDIUM:
-cell with targeted insertion
- cell with random insertion
- cell with no insertion - Cells carrying Targeted mutation (pickout on medium)
ES Cells KO Mouse Production
- Normal Chromosome:
with targeted mutation - ES cells from brown mouse : A/A:M/M
- INTO BLACK; a/a; M/M FEMALE BLASTOCYST-STAGE EMBRYO
- a/a;M/M plus A/A; M/m
= ALTERED EMBRYO - EMBRYO IN SURROGATE MOTHER
- NEWBORN CHIMERIC MALE (CARRY CELLS FROM 2 MOUSE STRAINS)
Why are KO (knockout mice) great? = 2
- led to important discoveries of gene function in vivo
- produced useful models of human disease
Problems with Knockouts = 5
- Embryonic Lethal
- Developmental Abnormalities
- Strain Effects
- Altered Expression of Other Genes
- No Phenotype
