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”)