• Used magnetic particles to improve delivery of
Biomolecules, including DNA, into cells.
• protein production for clinical or research applications
• changing the protein expression profile of a cell to
assay for the effects of a gene viz.
Over expression of gene
RNAi-reduction in expression of gene
• addition of genetic markers to a cell line
The process of transferring foreign DNA
fragments into a recipient (host) cell for
growth and replication.
Bacteria must be “competent" for the uptake
of foreign DNA fragments.
◦ produce specialized proteins◦ produce specialized proteins
◦ competence may be triggered heat shock or CaCl2
DNA segments must be small
◦ ~ 500 kb
1. The plasmid vector must be cut with a restriction
endonuclease (aka: restriction enzyme).
2. DNA ligase joins the DNA fragment & vector DNA
3. Host cell is made competent so that plasmid can
4. Transformed cells are grown on selection media .
5. The transformation system includes 3
components viz. mode of delivery, selectable
marker and propagation of clones.
Cold Shock with high concentrations of
calcium chloride and magnesium chloride make
the cells competent.
For preparation of comp cells..For preparation of comp cells..For preparation of comp cells..For preparation of comp cells..
Grow cells in SOB medium
1. Chemical treatmentChemical treatmentChemical treatmentChemical treatment
Grow cells in SOB medium
Cells treated with high conc.of chemicals
Immediate cold shock at -1960C.
store at -800C and use for transformation
The bacterial cells are taken into the
mouse and given shock of 2.5 mV.
Electric Shock Opens Pores in Cell
Shoots projectiles of gold or tungsten coated
with DNA or RNA into cells
Generally used with Eukaryotes
Incase of plants it is either
Non Integrative:- in it the transgene is maintained stably in an
extrachromosomal state but not transfer throughout the generation.
e.g.:- virus vectore.g.:- virus vector
Integrative:- in it the foreign gene integrated into the host gene and so can
pass onto the next generation.
A variety of process used to introduced transgene into plant cells,
Can be grouped into 2 categories:-
1.Indirect gene transfer
2.Direct gene transfer
Gene transfer through Agrobacterium.
These occur in 2 ways:-
a. co-culture with tissue explants
b. In plant transformation
By placing viral genome into the T-DNA of a Ti-plasmid
Agrobacterium used for co-culture with the plant cell
Used with such viruses which have to be transmitted by an insect vector
Used for successful infection.Used for successful infection.
Maize RS streak virus
Wheat dwarf virus
Introduction of foreign DNA without involvement of a biological agent.
Various methods are as follows:-
a> chemical methodchemical methodchemical methodchemical method:- by using certain chemicals.
b> electroporationelectroporationelectroporationelectroporation:- Introduction of DNA into cell by exposing them for some
period to electrical pulses.period to electrical pulses.
.it induce transient pores in the plasma membrane.
Basically 2 systems are there-
1.Low voltage-long pulse method- produces transient transformation
2.High voltage-short pulse method- produces stable transformation
c> Particle Gun MethodParticle Gun MethodParticle Gun MethodParticle Gun Method -1-2micro m tungsten & gold particle DNA to be
LASER−INDUCED DNA DELIVERY
It is used to deliver DNA into plant cell.
Lasers puncture transient holes in the cell
Used for high transfection freequency (10 3).Used for high transfection freequency (10 3).
There is little information on transient
expression or stable integration.
Gene cloning –T.A.Brown
Genes and Genome- Richard J. Reece.