L10 Transformation in multicellular organisms Flashcards
Yeast summary
- rapid, easy, range of vectors
- large numbers of transformants means cloning by complementation is possible
- homologous integration or autonomous replication
- gene inactivation or modification available
Aspergillus summary
- rapid, easy
- cloning by complementation
- non-homologous and homologous (nkuAdelta)
Multicellular organisms
- only certain cells contribute to the germ line
- to generate a fully transgenic organism carrying introduced DNA in every cell, you need a mean to alter the germ line
Plant transformation
- electroporation of plant cell protoplasts
- biolistics “gene gun” and tungsten beads
- Agrobacterium mediated transformation: uses Ti (tumour inducing) plasmid from Agrobacterium tumefaciens to promote integration of DNA
Protoplast
Protoplast = the protoplasm of a living plant or bacterial cell whose cell wall has been removed
Gene gun
Gene gun = shoot microscopic metal beads coated with DNA at plant tissue to penetrate through and be taken up by the cell
Crown gall disease
- caused by Agrobacterium tumefaciens
Lead to massive proliferation of cells which creates the tumour
Growth of tumour cuts of nutrient flow, starving the plant of its nutrients, ultimately leads to death
Agrobacterium mediated transformation of plant cells
See OneNote diagram
- crown gall formation
- vir genes: virulence genes
- T-DNA
Virulence genes = allow bacterium to infect and to transfer T-DNA to plant cell and to integrate T-DNA into plant genome
Tumour-inducing (Ti) plasmid
See OneNote diagram
Whatever is between T-DNA border region will be integrated into plant genome - useful transformation system
Insert/replace T-DNA with gene of interest and selectable marker flanked by T-DNA borders
Binary plasmid vectors
See OneNote diagram
- can be engineered very easily
- one plasmid has the virulence genes, the other has the T-DNA borders
Advantages:
Compared with co-integrated vectors, binary vectors present some advantages:
No recombination process takes place between the molecules involved.
Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium.
“Transient” Agrobacterium-mediated transformation by leaf infiltration
See OneNote
Agrobacterium-mediated transformation of the germline
See OneNote diagram
- engineered Agrobacterium culture
- transformation
- callus - cultured on selection media
- plant hormones
- transgenic plant
Callus
undifferentiated plant cells
Callus transformation
- Can inoculate callus with the gene you want
Floral dip
See OneNote
- Dip flowers into engineered agrobacterium culture, getting agrobacterium into the ovules which becomes seeds once they are fertilised
- Flowers ARE the germline
- Works really well with arabidopsis
- Proportion of seeds will be transformant , will be heterozygous
Selection of Arabidoptsis transformants
See OneNote
- germinate seeds on selection (herbicide or antibiotic)
T1 = heterozygous for T-DNA, resistant (transgenic plants) T2 = 25% homozygous for T-DNA
Uses for plant transformation - T-DNA library
T-DNA library
- integration of T-DNA is non-homologous so targeted inactivation is not possible
BUT
- random T-DNA insertion can disrupt gene function => can use inverse PCR to map T-DNA locations as we know sequence of that T-DNA
Inverse PCR
See OneNote Inverse PCR page
Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence.
Uses for plant transformation - Complementation
See OneNote diagrams
- introduce a copy of a gene to complement a mutant phenotype
If we want to prove that a mutation is in a particular gene
Look for rescue/complementation
- reporter constructs
Arabidopsis gene transfer summary
See OneNote summary page
Drosophila melanogaster transformation
- embryo
- micro-inject DNA into posterior pole (site of gonad development)
- requires P-element vector otherwise no transformants
Hybrid dysgenesis
See OneNote diagram
When P males are crossed to M females, the P elements in the male genome are mobilised
P-elements
- transposons
- inactive in somatic cells but have active transposase in germline cells
P element is an autonomous transposon, it include a transposase genes flanked by inverted repeats
Random insertion of transposons => deleterious mutations that affects viability
Engineer P-element for Drosophila transformation
See OneNote
Selection for Drosophila transformants
Screen for G0 flies which are mosaic - some of the cells were successfully transformed
Cells that have been transformed will become red, not all cells in that eye will have been transformed
G0 = mosaic flies G1 = red eyed flies are heterozygous for transgene G2 = 25% homozygous for transgene
Gene transfer in Drosophila summary
See OneNote summary page
Enhancer trap
See OneNote and enhancer trap page
- exploits non-homologous integration to identify new genes of interest
- If p-element lands next to an enhancer, it will activate that reporter gene
- can map insertion by inverse PCR
GAL4 Enhancer Trap
See OneNote
- use enhancer to drive tissue-specific expression
Gene Targeting by Homologous Recombination in Drosophila
See OneNote diagram
Integration of DNA by homologous recombination does not occur in Drosophila, which makes gene targeting difficult
FRT in the presence of flippase loops out into a circle, creates circle containing WT copy of the yellow gene
The restriction site creates a cut circle, DS break. DS break promotes homologous recombination at yellow locus.
BUT not as successful for other loci
RNAi
- RNA interference
- used to knock down gene expression
- widely used in non-model insects, plants, mammalian cells
- injection of double stranded RNA => specific ablation (removal) of corresponding endogenous mRNA
RNAi mechanism
See OneNote diagram
dsRNA cut up by dicer into siRNA
Loaded as ssRNA into RISC protein
RISC protein targets to complementary mRNA and cleaves it
dsRNAi in vivo to inactivate gene function
See OneNote diagram
- sense/antisense designed as complementary to gene of interest
Expression of dsRNA can be controlled
E.g. gene silencing
- inducible RNAi: heat-shock promoter
- tissue/stage specific RNAi: UAS (GAL4 inducible)
Drosophila gene transfer summary
- integration by P-element transposase, not by homology
- frequency too low for cloning by complementation
- can introduce any sequence e.g. reporter genes, enhancer trap
- gene inactivation difficult, knock down by RNAi
