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Molecular Genetics > Transgenic 1 > Flashcards

Transgenic 1 Flashcards

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1
Q

What is a Transgene?

A
  1. Gene/genetic material transferred from one organism to another (naturally or using genetic engineering) – generally germ line
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2
Q

A TRANSGENE May change the phenotype of the
organism its introduced into.

New gene may: 2

A

– retain the ability to produce RNA/protein

– alter the normal function of the t/g organism’s genetic code

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3
Q

WHY MAKE A TRANSGENIC ANIMAL? 5

A

1 *Identification of gene function

2 *Generation of animal models of diseases

3 *Drug validation

4 *Cell and organ research

  1. Others
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4
Q

Improvement of livestock WAS limited by?

NOW WHAT HAPPENS?

A

Improvement of livestock -traditionally limited by NATURAL VARIATION…

NOW- ‘make’ animals - express a trait of interest …

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5
Q

What are the 3 basic techniques to produce TRANSGENIC MICE:

A

(1) Pronuclei injection

(2) Retroviral gene transfer

(3) Embryonic stem cells - gene target

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6
Q

Transgenesis - history …

A
  1. 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.
  2. Gordon and Ruddle, 1980
    mouse born with genetic material injected into
    pronuclei of a fertilised mouse egg
  3. The term “transgenics” was born …

TRANSGENE IS THE INTRODUCED DNA…

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7
Q

why is mouse USED AS THE MODEL? =7

A

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

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8
Q

What can FOREIGN DNA be used for in TRANSGENESIS? 5

A

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)

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9
Q

What is gene locus?

A

Gene Locus: Includes both the promoter and the transcription unit!

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10
Q

Parts of a Transgene - Recombinant DNA

A

PROMOTER
1. Distal (>100kb)

  1. Proximal (approx. 1kb)

TRANSCRIPTION UNIT:
- Coding and noncoding sequences (from 1kb to >200kb)

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11
Q

Promorter/enhancer sequences drive expression?
4

A

Promoter/enhancer sequences
….drive expression …
- tissue
- time
- amount
- control via regulatory elements

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12
Q

How are TRANSGENES DEVELOPED AND INCORPORATED?

A

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

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13
Q

Transgenic Technology
- PROMOTER VS cDNA examples

A

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
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14
Q

What is the USE OF REPORTER GENES? HOW (4)

EXAMPLES OF VISUALLY IDENTIFIABLE ONES: 3

A

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…

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15
Q

Genetically altered Mice:

KNOCKOUTS…
What is the in vivo function of a gene? -

A

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)

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16
Q

Pronuclei Injection
WHAT IS IT? PURPOSE? 3

A
  1. Introduce foreign DNA (the transgene) into the pronucleus (nucleus of a sperm/egg cell during fertilization)
  2. Get random incorporation of the DNA = ECTOPIC INSERTION
  3. Hopefully ~10-40% of injected pronuclei contain construct transgene will be expressed in the germline & generate a strain of animals expressing the desired gene
17
Q

Pronuclei Injection DNA is… 3

A

DNA
1. very clean

  1. no vector sequences
  2. promoter - reporter
18
Q

Pronuclei Injection METHOD?
How many mice and why do need them?

A

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

19
Q

GENERATING A TRANSGENIC MOUSE: How does it work..is it due to amount of DNA or expression

A
  • 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)
20
Q

STEPS IN GENERATING A TRANSGENIC MOUSE? 5

A
  1. inject foreign DNA into one of the pronuclei.
  2. Fertilised mouse egg prior to fusion of male and female pronuclei.
  3. Transfer injected eggs into foster mother
  4. About 10 to 30% of offspring contains injected foreign DNA. Foreign DNA is present in equal amounts in all tissues.
  5. Mice expressing foreign DNA are BRED to continue the DNA in germ line.
21
Q

How is transgenic mouse identified?

  • labs
A

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.

22
Q

Disadvantages of pronuclei injection: 3

A

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

23
Q

Embryonic stem cells overcome the problems of…

A

Pronuclei Injection

24
Q

What are EMBRYONIC CELLS?

A

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)

  1. 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

  1. genes can be introduced into ES cells via transfection,
    retroviral infection, injection, electroporation
25
Q

CHIMERA?

A

Chimera = organism composed of tissues containing two or more genetically distinct cell types

26
Q

Generating a KO mouse
- producing the ES Cell

A
  1. MORULA:
    8 cells = 2 1/2 days
  2. MORULA
    16 cells = 3 days
  3. SELECTION OF BLASTOCYST (inner cel mass, blastocoel, Trophectoderm)
  4. in a CONTAINER = EMBRYONIC SREM CELLS + IRRADIATED FEEDER CELLS
27
Q

THE ADAVANTAGES AND JOBS OF EMBRPNIC CELLS?

A
  • Can direct transgene to endogenous gene = gene targeting/homologous recombination
  • can check recombinants before ‘making’ mice
28
Q

EMBRYONIC CELLS HAVE Two distinct advantages over embryos …

A
  1. SINGLE copy of transgene inserted
  2. identify clones expressing transgene prior to
    producing animals
29
Q

Targeting Constructs for Knockouts PURPOSE OF NEO

A

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.

30
Q

Targeting Constructs for Knockouts PURPOSE OF THYMIDINE KINASE (TK)

A

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

31
Q

ES CELLS - GENE KNOCK OUT:
PRODUCTION OF ES CELLS WITH A GENE KNOCKOUT

A
  1. Closed gene: with Exon 1 and 2
  2. ADD tk+ GENE
    INSERT neo^R into EXON 2
  3. Targeting vector
    tk+
    NEO^R
  4. ADDING TARGETING VECTOR
  5. CULTURED MOUSE EMBRYONIC STEM CELLS
32
Q

ES Cells - Targeted insertion
Homologous Recombination:

POSSIBLE OUTCOMES

A
  1. Vector
    Target gene in chromosome
    –> neo^R tk-
    Chromosome with targeted insertion
  2. Ectopic random insertion
    Vector
    —>non-target gene in chromosome
    tk+ neo^R
    Chromosome with random insertion
  3. no insertion
    vector
    nontargt gene in chromsome
    –>
    Unchanged chromosome
    tk- neo^s
33
Q

Embryonic cell SELECTION
- Selection of cells with gene knockout

A
  1. Neomycin analog = kills neo^S cells + Ganciclovir = kills tk+ cells
  2. —-> Add to MEDIUM
  3. in MEDIUM:
    -cell with targeted insertion
    - cell with random insertion
    - cell with no insertion
  4. Cells carrying Targeted mutation (pickout on medium)
34
Q

ES Cells KO Mouse Production

A
  1. Normal Chromosome:
    with targeted mutation
    • ES cells from brown mouse : A/A:M/M
  3. INTO BLACK; a/a; M/M FEMALE BLASTOCYST-STAGE EMBRYO
  4. a/a;M/M plus A/A; M/m
    = ALTERED EMBRYO
  5. EMBRYO IN SURROGATE MOTHER
  6. NEWBORN CHIMERIC MALE (CARRY CELLS FROM 2 MOUSE STRAINS)
35
Q

Why are KO (knockout mice) great? = 2

A
  • led to important discoveries of gene function in vivo
  • produced useful models of human disease
36
Q

Problems with Knockouts = 5

A
  1. Embryonic Lethal
  2. Developmental Abnormalities
  3. Strain Effects
  4. Altered Expression of Other Genes
  5. No Phenotype

Molecular Genetics (21 decks)

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