Gene Transfer

소개글

Gene Transfer에 대한 보고서 자료입니다.

목차

1. Agrobacterium-mediated transformation

2. Particle Bombardment

3. Membrane permeabilization using chemicals

본문내용

VirE2. Transport of the T-complex to plant cells necessitates action of VirD4 and the 11 VirB proteins, and is probably driven by ATP hydrolysis. VirB proteins may form a transmembrane pore that comes into contact with the plant cell. The nuclear-localization signals VirD2 and VirE2 mediate nuclear uptake of the T-complex by the nuclear pore complex(NPC). Finally, the T-DNA integrates into the plant genome, and expression of its encoded genes leads to the production of opines (nutrient for Agrobacterium) and hormones, causing tumor formation in the infected plant.
▶ Particle Bombardment
ㆍcan deliver DNA into virtually fo foreign genes
ㆍgenotype independent
ㆍbe used to study the transient expression of foreign genes
Agrobacterium의 host-range limitation
protoplast : whole plant regeneration
⇒ 문제점극복하기위한 방법
ㆍtungsten or gold microprojectiles (1~5㎛) DNA coating
▶ Membrane permeabilization using chemicals
ㆍpolyethylene glycol (PEG) or Calcium phosphate cause reversible permeabilization
of the plasma membrane
ㆍprotoplast (lack cell wall) + chemicals + naked DNA co-incubation
▶ Electrophoration
ㆍprotoplast + DNA + high-voltage electric fields
▶ Liposome mediated or microinjection
▶Regeneration of Transgenic Plant
1. Plant tissue culture
ㆍ Totipotency : unique ability of isolated plant cells to regenerate into a whole plant
ㆍ Callus formation - form almost any plant tissue using plant growth regulators
( ex-cytokinin, auxin)
ㆍ Protoplast generation
┌ form callus
│ form explants (leaves, roots, pollen, endosperm)
└ can be prepared either by mechanical or enzymatic removal of the cell wall
ㆍorganogenesis - shoot & root formation by separate, defined media
2. Somaclonal Variation
Plants regenerated by tissue culture may display a different to that of the plant
from which the cells originated
※원인
-ㆍchromosome rearrangements
-ㆍthe activation of endogenous transposons
-ㆍchanges in DNA methylation status
-ㆍmutation
※can be minimized
-ㆍexplant와 같이 semi-organized tissue를 사용한다면
-ㆍtarget cell이 즉시 혹은 callus induction의 짧은 기간 직후 embryogenesis로 들어갈 수 있다면
-ㆍDNA를 직접 germ line에 넣으면 된다.
▶ Selection and Identification of Transformed Cells
1. Selectable Marker
조건 : plant cell에 non-inhibitory
종류
① Neomycine phosphotransferase, typeⅡ(NPTⅡ)
- the most widely used selectable marker
- prokaryotic transposon Tn5에서 isolation
- kanamycine, G418(aminoglycoside compound) - phosphorylation시켜 detoxification
- this marker dose not work in all plant species. Arabidopsis thaliana
② Hygromycin phosphotransferase(hph)
③ Acetyltransferase - gentamycin resistance
④ Dihydrofolate reductase (DHFR)
- DHFR은 methotrexate에 의해 blocking
- mutant mouse에서 methotrexate에 대해 500배 정도 낮은 affinity를 지닌
DHFR을 구해 CaMV 35S promoter에 fusion 시켜 methotrexate reisistant
⑤ Herbicide reisistance
⑥ GUS activity
β-glucuronidase
X-glucuronide
-------------------->
blue color
⑦ CAT activity 14C chloramphenicol
⑧ Luciferase
luciferase
luciferin + ATP
-------------------->
oxidizes luciferin + AMP + CO2 + light

키워드

Transferred T-DNA ,   Gene Transfer,   Agrobacterium-mediated transformation