13-3 Cell Transformation13-3 Cell Transformation13-3 Cell Transformation13-3 Cell Transformation

(Changes in Cell(Changes in Cell’’s DNA)s DNA)

Review

• Which enzyme is used as a scissor in genetic engineering ? Which one is used as glue?

• What’s the function of gel electrophoresis?

Sciessor: Restriction EnzymeGlue: DNA ligase

Separate DNA fragments by their different size

13-3 Cell transformation

• Cell transformation= change the cell’s DNA

• A cell takes DNA from outside. The external DNA becomes part of the cell’s DNA

1) Transforming bacteria cells– Using plasmid

• small circle of extra bacterial DNA, double strand

• Foreign DNA can be inserted into plasmid

– Forms recombinant plasmids

– Plasmids used in genetic engineering are called vectors.

– Can deliver DNA into another cell

Plasmid

1) Transforming bacteria cells– Using plasmid

• Plasmid has a origin site for replication

• Plasmid has a genetic marker (usually a gene for resistance to antibiotics)

• two types of plasmid integration into a host bacteria

Restriction enzyme cuts molecule of chromosomal DNA or cDNA

recombinant plasmids containing foreign DNA

host cells containing recombinant plasmids

Same enzyme cuts same sequence in plasmid DNA

Foreign DNA, plasmid DNA, and modificationenzymes are mixed

DNA or cDNAfragments with sticky ends

plasmid DNA with sticky ends

What if insertion eukaryotic DNA into bacteria ?

Will there be any problems ???

Transcription

Pre-RNAPre-RNA mRNAmRNADNADNA

RNA polymerase

No introns

mRNA splicing

Bacteria don’t know how to deal with introns

USE cDNA!!!

mRNA transcript

mRNA–cDNA hybrid

single-stranded cDNA

double-stranded cDNA

Reverse transcription= Making cDNA

cDNA -- A DNA reverse transcription from a matured mRNA

Reverse transcriptase

DNA polymerase

“c” -- complementary `

2) Transforming plant cells

• Recombinant plasmid is transformed to plant– By using bacterium Agropbacterium

tumefaciens– By gene gun injection

By using Agrobacterium

Plasmids carrying the geneof interest is transferred

to agrobacterium

The agrobacterium inserts the foreign DNA into the plant cell to form transgenic cell

By using gene gun

To coat metal particleswith gene containing

plasmids

Gene gun injects the DNA covered particles into the plant cell

3) Transforming animal cells

• Animal cell

– Inject into egg nucleus some enzymes may help to insert

foreign DNA into chromosomes– Gene replacement ( gene “knockout”)