Lecture 23 Flashcards Preview

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Flashcards in Lecture 23 Deck (21):
1

Multicellular organisms:

- Only certain cells contribute to the germ line in multicellular organisms
- To generate a fully transgenic organism carrying the introduced DNA in every cell, you need a means to alter the germ line

2

Plant transformation:

- Electroporation of plants cell protoplasts
- Biolistics 'gene gun' and tungsten beads
- Agrobacterium mediated transformation, using a Ti (tumor inducing) plasmid from agro, to promote integration of DNA

3

Agrobacterium mediated transformation of plant cells:

- Ti plasmids of agrobacterium inserted into plant cells. Ti plasmids get transferred into the plant
- Crown gall disease develops (over growth of cells), blocking xylem and phloem
- Agro transfers part of it's DNA into the plant DNA

4

Ti plasmid (unengineered):

- It's own oriV
- Virulence genes (encode proteins required for infection, T-DNA transfer and integration into host genome)
- T-DNA genes (encode proteins required for gall formation, this material is inserted into the plant genome)

5

Ti plasmid vector:

- Insert/replace T-DNA with the gene of interest and selectable marker flanked by T-DNA borders

6

Binary plasmid vectors:

- Create two separate plasmids
- One has virulence genes, for infection and mobilisation
- The other has the genes of interest flanked by the T-DNA boarders, critical for mobilisation

7

Infecting the plant with Agrobacterium carrying the T-RNA construct

- Infect callus tissue which is totipotent
- Floral dip method, just dip/paint the base of the stem
- Genes will be inserted

8

Which cells will take up the T-DNA plasmid?

- Random!
- Look for tissue developing into seeds (germ line), not vegetative
- Screen collected seeds for those that have taken up the selectable marker (drug resistance etc)
- 1/1000 seeds will contain T-DNA, which seems low but is actually quite feasible
- This will create heterozygous plants, which can be crossed to create homozygotes

9

Plant transformation:

- Integration via T-DNA flanks, not homology, so is non-homologous
- Used to confirm gene identify, probably not for cloning genes by complementation
- Introduce reporter constructs, such as GUS or lacZ

10

Drosphila melanogaster transformation:

- To introduce DNA into the embryo micro-inject DNA into the posterior pole (which is the site of gonad development)
- This will only work if a P-element vector is used, as dros are very resistant to introduction of DNA

11

Hybrid dysgenesis:

- Occurs wen certain combinations of strains are cross
- P strains contain a resident transposable elements, called the P element, M strains lack this element
- When P males are crossed to M females, the P elements in the male genome are mobilised and reduced fertility and elevated rates of mutations occurs

12

P-elements:

- Encode a transposase that can result in excision of the resident P-elements and re-insertion into random sites within the Drosophila genome
- The transposase contains inverted repeats which allow mobilisation

13

Dros transformation using P-elements:

- Inject DNA into M (w- or rosy-) embryo with no P-elements present
- The DNA will have the genes of interest, a selectable marker, flanked by inverted repeats.
- On a second plasmid insert a transposase with no inverted repeats
- This mobilises the DNA between the inverted repeats

14

Creating homozygous drosophila:

- DNA integration is via repeats and transposase
- This is non-homologous
- Only some cells will have taken up the DNA: mosaics
- Cross the mosaics and look for selectable marker phenotype (often white)
- Cross mosaics with white eyes, produces heterozygotes

15

Gene transfer in drosophila:

- Requires P-element vector, inject dNA into M-type embryo and take moaics to produce G1 then G2
- Low numbers
- DNA will be integrated non-homologously
- Used to introduce cloned genes to confirm and assess the effect of a mutation on gene function in vivo and monitor reporter gene expression

16

Enhancer trap:

- Uses non-homologous integration to identify new genes of interest
- Reporter gene (lacZ) is fused to a minimal promoter with no regulation and flanked by inverted repeats, and introduced to the organism
- Dros transformants are screened for regulated expression of lacZ
- If it is integrate near the gene of interest the insertion provides a tag for cloning!

17

Gene targeting in dros:

- Non-random, non-homolgous
- DNA introduced by the P-elements, so integrates randomly
- FLP/FRT recombinase generates plasmid in vivo
- Plasmid is cut by rare RE to increase the frequency of homologous events
- Induce FLIP recombinase, and recombination between FRT sequences generates a plasmid
- Plasmid cut by RE inside the nucleus
- Homologous recombination occurs
- Could be applied to any gene, but is tooooo hard!

18

RNAi for targeted inactivation of gene function:

- used to knock down gene expression
- Injection do dsRNA, specific ablation of corresponding endogenous mRNA
- So can use this biological system to bring about RNAi type inactivation of gene function in the organism
- Create constructs expression siRNA in vivo, containing promoter, sense and antisense version of gene of interest
- Generate dsRNA inside the cell, DICER will chop it up, and RNAi will start

19

attP/attB:

- Targeting incoming DNA to known locations, the attP/attB sites

20

dsRNA expression can be controlled:

- Inducible at the tissue or stage specific mRNA level
- A promoter only active at a certain tissue or stage of development
- Allows specific knock down of the gene of interest

21

To clone a gene by complementation:

- We need to know if the mutation is recessive or dominant
- This is because we need to know if it is cloning by complementation
- Detecting WT version of gene is to see a restoration of the WT copy
- If the mutant was a dominant, you would no see rescue, so you would not be able to pick up complimentation