L10 Transformation in multicellular organisms

Question 1

Yeast summary

Answer 1

• 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

Question 2

Aspergillus summary

Answer 2

• rapid, easy
• cloning by complementation
• non-homologous and homologous (nkuAdelta)

Question 3

Multicellular organisms

Answer 3

• 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

Question 4

Plant transformation

Answer 4

1. electroporation of plant cell protoplasts
2. biolistics “gene gun” and tungsten beads
3. Agrobacterium mediated transformation: uses Ti (tumour inducing) plasmid from Agrobacterium tumefaciens to promote integration of DNA

Question 5

Protoplast

Answer 5

Protoplast = the protoplasm of a living plant or bacterial cell whose cell wall has been removed

Question 6

Gene gun

Answer 6

Gene gun = shoot microscopic metal beads coated with DNA at plant tissue to penetrate through and be taken up by the cell

Question 7

Crown gall disease

Answer 7

• 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

Question 8

Agrobacterium mediated transformation of plant cells

Answer 8

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

Question 9

Tumour-inducing (Ti) plasmid

Answer 9

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

Question 10

Binary plasmid vectors

Answer 10

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.

Question 11

“Transient” Agrobacterium-mediated transformation by leaf infiltration

Answer 11

See OneNote

Question 12

Agrobacterium-mediated transformation of the germline

Answer 12

See OneNote diagram

1. engineered Agrobacterium culture
2. transformation
3. callus - cultured on selection media
4. plant hormones
5. transgenic plant

Question 13

Callus

Answer 13

undifferentiated plant cells

Callus transformation
- Can inoculate callus with the gene you want

Question 14

Floral dip

Answer 14

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

Question 15

Selection of Arabidoptsis transformants

Answer 15

See OneNote

• germinate seeds on selection (herbicide or antibiotic)

T1 = heterozygous for T-DNA, resistant (transgenic plants)
T2 = 25% homozygous for T-DNA

Question 16

Uses for plant transformation - T-DNA library

Answer 16

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

Question 17

Inverse PCR

Answer 17

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.

Question 18

Uses for plant transformation - Complementation

Answer 18

See OneNote diagrams

1. 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

2. reporter constructs

Question 19

Arabidopsis gene transfer summary

Answer 19

See OneNote summary page

Question 20

Drosophila melanogaster transformation

Answer 20

• embryo
• micro-inject DNA into posterior pole (site of gonad development)
• requires P-element vector otherwise no transformants

Question 21

Hybrid dysgenesis

Answer 21

See OneNote diagram

When P males are crossed to M females, the P elements in the male genome are mobilised

Question 22

P-elements

Answer 22

• 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

Question 23

Engineer P-element for Drosophila transformation

Answer 23

See OneNote

Question 24

Selection for Drosophila transformants

Answer 24

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

Question 25

Gene transfer in Drosophila summary

Answer 25

See OneNote summary page

Question 26

Enhancer trap

Answer 26

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

Question 27

GAL4 Enhancer Trap

Answer 27

See OneNote

- use enhancer to drive tissue-specific expression

Question 28

Gene Targeting by Homologous Recombination in Drosophila

Answer 28

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

Question 29

RNAi

Answer 29

• 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

Question 30

RNAi mechanism

Answer 30

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

Question 31

dsRNAi in vivo to inactivate gene function

Answer 31

See OneNote diagram

• sense/antisense designed as complementary to gene of interest

Question 32

Expression of dsRNA can be controlled

Answer 32

E.g. gene silencing

• inducible RNAi: heat-shock promoter
• tissue/stage specific RNAi: UAS (GAL4 inducible)

Question 33

Drosophila gene transfer summary

Answer 33

• 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