1. Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

2. Method of Introducing Transgene in Mouse

3. Herpes thymidine kinase gene

4. There are 3 possible fates. Each possibility produces different results. Targeted insertion results in cells that are neomycin- resistant and do not harbor the tk gene. Cells can be isolated selectively.

5. When cells are grown in the presence of neomycin analog and ganciclovir, only cells with targeted insertion will survive. Such cells can be cultured and introduced into embryos in the blastocyst stage.

6. Generation of Knockout Mice ES cells from agouti mouse (A/A) with targeted insertion of a mutant allele are inserted into blastocyst-stage embryo of a/a genotype. Embryo is inserted into a surrogate mother and progeny that are chimeric for coat color are selected.

7. Generation of Knockout Mice Chimeric male is mated to a/a female. DNA from progeny are screened for targeted insertion. A/a mice with targeted insertion are crossed. Progeny from this cross are screened for targeted insertion and phenotype(s) of mice that are homozygous for the insertion are scored.

8. Gene Therapy in Mammals

9. Human Gene Therapy Approaches to treatment of diseases Approaches to treatment of diseases Transfer of DNA into afflicted individual Transfer of DNA into afflicted individual 1990: First clinical trial. Severe Combined Immunodeficiency (SCID) - defective adenosine deaminase (ADA) gene. Afflicted patients express no functional T or B lymphocytes.

10. Delivery Approaches to Somatic Therapy Direct Administration: Direct Administration: Introduction of normal gene using a virus retrovirusadenovirus Indirect Administration: Indirect Administration: Reintroduction of cells made transgenic

11. Criteria for a Disease for Gene Therapy 1.Mendelian inheritance 2.One gene product for the gene 3.Knowledge of function of gene product 4.Availability of normal gene sequence 5.Localized symptoms for the disease 6.Availability of delivery system

12. ~ 25,000 genes in humans are potential candidates for gene therapy.

13. Cystic Fibrosis Disease is usually fatal. Lungs, other organs are clogged with mucus. repeated bouts of pneumonia heart stressed by decreased lung capacity delivery failure of pancreatic enzymes Life expectancy of 20-25 years ~1 of 29 Caucasians are carriers ~1 of 1600 Caucasians are affected ~30,000 Americans are affected

14. Cystic Fibrosis Caused by recessive mutation (chromosome 7) Caused by recessive mutation (chromosome 7) Failure to pump Cl- ions across cell membrane Failure to pump Cl- ions across cell membrane Missing Cl- ion channel protein (cystic fibrosis Missing Cl- ion channel protein (cystic fibrosis transmembrane protein or CFTR) Normal gene sequence available (cloned in 1989) Normal gene sequence available (cloned in 1989) Normal gene 250 kb in size Normal gene 250 kb in size Transcript of 6 kb in size Transcript of 6 kb in size Gene product of 1480 amino acids in length Gene product of 1480 amino acids in length Over 1500 mutations documented Over 1500 mutations documented

15. Cystic Fibrosis as Candidate for Gene Therapy 1.Recessive gene on chromosome 7. 2.CFTR is the gene product. 3.CFTR is an ion channel protein. 4.Normal gene sequence has been cloned. 5.Symptoms are localized to specific organs. 6.Adenovirus (cold virus) can be used to deliver normal sequence to lung cells.

16. Human DNA Information: Ethical Concerns Who should have access to your DNA information?