Dolly the sheep ( ) 1. Animal and human cloning 2. Gene cloning

1. Animal and human cloning

To "clone a gene" is to make multiple copies of it in vivo What is Gene cloning?

Why Clone DNA? A particular gene can be isolated and its nucleotide sequence determined Protein/enzyme/RNA function can be investigated Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes, e.g. insulin production

Insulin overexpression in bacteria Gene encoding insulin Ligation RE1 RE2 RE1 RE2 RE1 RE2

Insulin overexpression in bacteria Transformation into E. coli cells Insulin

What do we need for gene cloning? Insert Plasmid vector ligation transformation Target DNA Ligation of the palsmid+Insert Plasmid Vector transformation

Plasmids are Used to Replicate a Recombinant DNA Plasmids are small circles of DNA found in bacteria. Plasmids replicate independently of the bacterial chromosome. Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid.

All plasmids contain: 1. Origin of replication (ori) 2. Selection marker (Ampicillin) 3. Multiple Cloning Site (MCS) Plasmid vectors Circular DNA molecules capable of autonomous replication in living cells A Plasmid (vector) is a DNA molecule used for transferring foreign DNA fragments (genes) into host cells MCS

Insert – Target DNA 2. Restriction Enzymes 1. PCR product RE1 RE2 RE1 RE2 T T

Ligation REs will produce ends that enable the gene to be spliced into a plasmid Ligation

REs and DNA ligase DNA ligase Ligation of the insert to the plasmid cut with only one enzyme

RE1 RE2 Ligation of the insert to the plasmid cut with only two enzyme ligation There is two possible outcomes

Transformation Two main methods: 1.Chemical transformation – Chilling cells in the presence of Ca2+ prepares the cell walls to become permeable to plasmid DNA. Cells are briefly heat shocked which causes the DNA to enter the cell 2.Electoporation- making holes in bacterial cells, by briefly shocking them with an electric field of kV/cm. Plasmid DNA can enter the cell through these holes. Use of bacterial cells to amplify the DNA of interest

Possible products of the transformation: Plasmid + insert Ampicillin resistant Plasmid without insert Ampicillin resistant No plasmid No ampicillin resistance How can we differentiate between the bacteria containing plasmid+insert and the ones with the self ligated plasmid (no insert)? Plasmid + insert Ampicillin resistant

Our lab experimemet Insert that was amplified by PCR pGEM Vector ligation Transformation Screening

Possible products of the transformation: Plasmid + insert Ampicillin resistant Plasmid without insert Ampicillin resistant No plasmid No ampicillin resistance How can we differentiate between the bacteria containing plasmid+insert and the ones with the self ligated plasmid (no insert)? Plasmid + insert Ampicillin resistant

Cloning procedure + IPTG + X-Gal transformation

Lac Z gene promotor RNA pol. Gene expression dogma DNA LacZ mRNA Ribosome β-galactosidase RNA Protein X-gal BLUE colonies WHITE colonies X-gal

LacZ promotor operator Repressor Lac Z gene LacZ promotor RNA pol. IPTG LacZ promotor operator IPTG RNA pol. IPTG X-gal Β-galactosidase X + galactose BLUE colonies Cells which produce ß-galactosidase form BLUE colonies. WHITE colonies Cells without ß-galactosidase production form WHITE colonies.

X XX X X Plasmid without Insert Plasmid +Insert without plasmid Screening LacZ pGEM Insert WHITE colonies BLUE colonies promotoroperator T T

A plasmid DNA will be purified from the bacteria cells. Insert Vector Confirmation by digestion with restriction enzyme and separation of the digestion products on agarose gel EcoRI Plasmid DNA will be digested with EcoRI, and analyzed by gel electrophoresis for identification of the clone containing insert. pGEM