1. Recombinant DNA Technology
Site directed mutagenesis
Genetics vs. Reverse Genetics
Gene expression in bacteria and viruses
Gene expression in yeast
Genetic engineering in plants
Genetic engineering in animals
Gene therapy in mammals
Detection of disease alleles

3. Recombinant DNA Technology
Site directed mutagenesis
Base pair substitution
Insertion
Deletion
Plasmid or PCR based approach

9. Genetics
Mutant phenotype
Mutant allele
DNA sequence
Protein sequence

10. Reverse Genetics Protein sequence DNA sequence Mutant allele
Mutant phenotype

11. Recombinant DNA Technology
Restriction fragment length polymorphism
Restriction digest
Gel or gel + Southern blot
Identify linkage to the mutant allele

13. Recombinant DNA Technology
Gene disruption
Gene knockout
Insert selectable marker into gene

16. Recombinant DNA Technology
Two-step expression vector
Chromosomal lac inducible T7 RNA pol
T7 RNA pol initiates expression of plasmid

18. Recombinant DNA Technology
Bacterial expression of hGH
No human signal sequence
Add bacterial signal sequence

21. Recombinant DNA Technology
Engineered bacteria
Synthesize new antibiotics
Actinorhodin (blue)
Medermycin (brown)
Hybrid mederrhodin A (reddish purple)

25. Recombinant DNA Technology
Baculovirus expression
150 kb genome.
Insect cells.
Low frequency recombination inserts foreign gene into viral genome.
Recombinant virus do not have coat protein.

28. Recombinant DNA Technology
Yeast
Vectors
Transformation
Gne regulation

32. Recombinant DNA Technology
Genetic engineering in plants
Ti plasmid
Agrobacterium tumefaciens
Crown gall disease
Genetically engineered plants

41. Recombinant DNA Technology
Transgenic Drosophila
Bacterial beta galactosidase gene
P elements

43. Recombinant DNA Technology
Transgenic sheep
Beta lactobumin promoter
Active only in mammary tissue
Microinjection into ovum
Transplant and test by PCR
High levels of transgene expressed in milk

45. Recombinant DNA Technology
Rodent gene knockouts
Create targeting vector
Homologous recombination in cells
Select for recombinants/knockouts G418 (Neo) eliminates wild type cells without vector integration and ganciclovir kills cells with randomly integrated vector (that carry the tk gene).

47. Recombinant DNA Technology
Rodent gene knockouts
ES cell knockouts

50. Recombinant DNA Technology
Transgenic rodents
Rat growth hormone responsive to heavy metals

53. Recombinant DNA Technology
Transgenic fish
Hormone complex with strong promoter in salmon

55. Recombinant DNA Technology
Gene Therapy
Injection of fertilized egg
Germinal therapy
Somatic therapy

57. Recombinant DNA Technology
Tracking disease alleles
Amniocentesis
Southern blotting
Common genetic disorders

61. Genomics Overview VNTRs Linkage RAPDs FISH and painting Genomes
Yeast two hybrid system
Microarrays

64. Genomics VNTRs Simple sequence repeats Highly variable
Excellent for linkage and human identity

69. Genomics
Linkage
Allele/marker segregates with a disorder

72. Genomics
RAPDs
Highly variable
Unknown origin
Reproducibility?

75. Genomics Chromosomal approaches FISH Paints
Study of large genomic alterations

78. Genomics Somatic cell hybrids Full complement of rodent chromosomes
One or more human chromosomes
Chromosome level PCR based mapping

80. Genomics
Genome sequences
Bacteria
Arabidopsis

83. Genomics Yeast two hybrid system Target and bait
Detect gene interaction

85. Genomics
Microarrays
High throughput
Genotyping
Gene expression