Gene Transfer

Question 1

How to create a transgenic plant?

Answer 1

1. Tissue culture. Need plant to regenerate from single cells.
2. Develop method to transform plant. Choose strain of agrobacterium. Select gene of interest to insert and selectable marker.
3. Grow on selective media so that only transgenic plants remain.
4. Include hormones on media(cytokines and auxin) as well as other enhancer (eg copper and zinc)to induce root and shoot uptake
5. Check plant actually has gene of interest

Question 2

Features of an ideal plant transformation system

Answer 2

1. High transformation efficiency
2. High co-transformation efficiency (want to be able to transform with more than 1 gene)
3. Precise transfer of desired genes only. Intact, single copy, transgene integration and no transfer of flanking vector sequence.
4. Low frequency of ‘collateral damage’
5. Broad applicability
6. Technical safety and simplicity

Question 3

Examples of selectable markers

Answer 3

aphA(nptII, neo) : antibiotic resistance to Kanamycin.

Bar(pat): herbicide resistance to Bialaphos.

Hpt(hyg): antibiotic resistance to hygromycin.

Question 4

Examples of reporter genes

Answer 4

Luc: light emissions

UID A( Gus) : colour change(blue)

Gap: fluorescent green under up light.

Question 5

Process of recalcitrance of plant cells

Answer 5

1. Callus induction- high auxin and moderate cytokinin
2. Shoot regeneration- high cytokinins, low/no auxin
3. Root induction- moderate auxin or no phytohormones

Question 6

Ways to confirm transgenicity?

Answer 6

Phenotype: appearance and assay and gene expression( northern, rt-qPCR, protein)

Genotype: PCR, southern blot( allows copy number estimate and ‘proves transgene insertion)

Question 7

What is totipotency?

Answer 7

The innate ability of a single cell to regenerate into a while, and reproducible organism.

Many plant cells have this

Question 8

What is floral pathway?

Answer 8

Involves injection of plasmid DNA with a hypodermic syringe into the developing inflorescence

Question 9

What is the viral pathway?

Answer 9

Make cDNA version of potyvirus, remove virulence sequence. Modify sequences for coat protein initiation and introduce a reporter gene.

Only achieved in tobacco and not very often that genome integration is achieved.

Question 10

Zinc Finger nucleases

Answer 10

ZFN cleavage induces a double-stranded break.

Imperfect repair of double stranded break causes target site mutation.

Homologous recombination with exogenous DNA creates a mutant allele.

Question 11

BIBAC?

Answer 11

Binary Bacterial Artificial Chromosomes

Recombination of BAC and binary vector leads to a split marker gene and A BAC inset with Agro left and right border sequences.

Allows for large insert libraries to be screened.
However large insets can be fragmented or rearranged. Allows identification of genome region with candidate gene but not actual candidate.

Question 12

TALENS?

Answer 12

Transcription activator like effect nucleases.

Artificial restriction enzymes made by fussing a DNA cleavage domain to a TAL effector DNA binding domain.
Binding domain consists of 33-34 conserved amino acids. 2 amino acids(6 nucletides) can be engineered to target specific sequence. Then introduced to cells to deliver double stranded DNA breaks.
DsDNA is then repaired by host cell.

Problem: if homologous not specific enough can lead to off target editing. Widely used in animals, less successful in plants

Question 13

CRISPR

Answer 13

Clustered regularly inter spaced short palindromic repeats