Polymerase chain reaction If DNA sequence is known, amplify specific gene directly

Cloning by PCR

humanDNA

RPE65gene

PCR

Cloning by PCR• Human DNA• RPE65-specific 20-nt primers• Taq DNA polymerase• dNTPs

5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG3′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC

part of RPE65

heat

5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG TTTGACACCT

5′primer

TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAAC

heat

5′ CCAGGTTGAG TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT

5′

primerCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA

Cloning by PCR

Once amplified, ligate and transform as before

Cloning by PCR

+amplified copiesof RPE65 gene

plasmidvector

Genetic engineering

Modified microorganisms: Insulin, growth hormone, clotting factors, EPO… HPV vaccine Ethanol from cellulose Oil-eating bacteria

Modified plants and animals BT corn Roundup-ready soybeans Golden rice

Modified humans Gene therapy

Genetic engineering

Previously derived from blood donations First “pharmed” drug approved by FDA (Feb 6, 2009)

Growing Human Antithrombin

Transgenic animals

Expression Vector

hAT

hAT

PCR

hAT

Transgenic animals

hAT

Transgenic animals ATryn 1 goat = 90,000 blood

donations

Transgenic plants Pest resistance Herbicide resistance Delayed ripening Increased nutritional values

Electroporation Agrobacterium tumefaciens

Transferring Genes to Plants

Human Gene Therapy

What diseases can we treat with Gene therapy?Replace genes, control expression

Microinjecting DNA into Cells

Transfection

Moving Genes around

hAT

DNA viruses: Adenovirus Retrovirus/Lentivirus

Moving Genes around

First therapy experiment: X-SCID (1990) First success: Metastatic melanoma (2006)

2 of 15 Patients had tumors shrink/disappear LCA2

Success?

Almost perfect… Adenovirus: elicits massive inflamation. Retrovirus: insertional mutagenesis

Embryonic Stem Cells Embryonic stem cells Embryonic germ cells

Induced Pluripotent stem cells Use of viral vectors Inject fibroblasts transduced with OCT4, SOX2, KLF4, and c-

MYC.

17.26

Will we cause new disease using GMOs? What is normal? Somatic vs. Germline therapy? Human cloning?

Ethical considerations