Transgenics 2 Flashcards
Understanding Conditional KOs?
= 4
- Cell type specific KOs and developmental-age
specific KOs can now be generated
2 * Site-specific recombinase technology – eg Cre-Lox.
Uses LoxP target sequences & enzyme Cre (a
recombinase) – derived from bacteriophage P1
3 * Activity of the Cre enzyme can be controlled –
expressed in a specific cell type or be triggered by
a specific external stimulus (chemical signal/heat)
4 * Other similar systems (eg FLP-FRT recombination
system) exist
How to Create a Conditional KO mouse?
1 -create a transgenic where the gene of interest is flanked by loxP sites, and another in which
the transgene is Cre driven by a specific promoter.
2 -If Cre is active,
catalyses a recombination event between the loxP sites
The Genetic Engineering of Plants -WHY? = 7
- Herbicide resistance
- Pest resistance
- Nutritional & flavour
improvement - Storage & marketability improvement
- Land care & rehabilitation
- Biofuels
- Basic science
Understanding Agrobacterium & the Ti Plasmid = 4
Agrobacterium tumefaciens
1 *soil bacterium
2 *causative agent of crown gall disease
3 *infects many diverse species, especially dicotyledons
(entry = wounds)
4 *contains the Ti (tumour inducing) plasmid
What is the Ti plasmid?
Ti plasmid:
*T-DNA (transfer DNA)
*flanked by 25 bp repeats
*encodes genes promoting tumour (gall) and bacterial growth
*vir region
* = encodes genes necessary for transfer of T-DNA
Integration of the Transgene into the Plant Genome
In an Agrobacterium cell within an infected plant:
1. *nick in one strand of Ti plasmid at one end of T-DNA
- *single-stranded DNA-binding protein (SSBP) binds
- *second nick at other end of T-DNA
- *replication replaces T-DNA region
5 *T-DNA transferred to plant cell nucleus & integrated into chromosome
*one or more copies of T- DNA inserted
*random insertion
*inherited in a Mendelian fashion
Binary Vector System
Separation of T-DNA & vir regions - easier manipulation.
Both plasmids required for transformation
Binary Vector System STEPS
A. T-DNA in Hybrid plasmid —-> Hybrid plasmid cut open by RESTRICTION ENZYMES –> Foreign DNA with sticky ends into RECOMBINANT PLASMID
B. RECOMBINANT PLASMID —> transferred into AGROBACTERIUM —> agrobacterium tumefaciens; Modified Ti plasmid containing vir gene but no T-DNA
C. Agrobacterium tumefaciens —> INFECTION of tobacco plants to Agrobacterium —> Transgenic tobacco plant —>
DNA = plant chromosomal; DNA, integrated T-DNA with foreign gene
Ti Plasmid T-DNA PROMOTERS ARE…3
1 - constitutive - continual
expression of downstream gene
2 - cell/tissue/organ-type specific - activated only in certain cells, tissues or organs
3 - inducible - activated by
chemical/environmental stimulus, resulting in expression of a downstream gene
Ti Plasmid T-DNA
RB AND LB?
RB - right border of T-DNA
LB - left border of T-DNA
Ti Plasmid T-DNA - ANTIBIOTIC RESISTANCE…
nptII – neomycin
phosphotransferase II gene,
confers kanamycin resistance
(kanr)
How to do Transgeneics in plants?
- Plant … discs removed from Tobacco leaf
- Leaf discs incubated with genetically engineered AGROBACTERIA for 24hrs
- Selection medium only allows plant cells that have acquired DNA from the bacteria to PROLIFERATE
- callus, SHOOT-INDUCING MEDIUM
- shoot (A) Transfer shoot-to- root inducing medium
- grow up rotted seeding
- adult plant carrying transgene that was originally present in bacteria.
What is BIOLISTICS?
the transgene dna is …4
Method of choice for monocotyledons (majority of important food crops)
Transgene DNA
1 *in T-DNA (e.g. between RB & LB)
2 *precipitated onto tungsten or gold particles (1 2 μm diameter)
3 *“shot” into plant cells
4 *particles enter nucleus, DNA integrated into genome
The Uses of Genetically Engineering Plants
- Functional Genomics
Forward Genetics
T-DNA insertion libraries of Arabidopsis thaliana
1 *T-DNA used to disrupt genes through insertional mutagenesis
2 *phenotypic screening for loss- and gain-of-function mutations
3 * eg defective flower, metabolism, defect in nutrient uptake
4 *disrupted genes identified by PCR, sequencing, & database searches
