1. Problem Results: Question: 1. You screen two libraries- cDNA; genomic
2. Clones are isolated having homology to PSY- 10 clones from each library
3. These are subcloned into pBluescript.
4. Protein expression is induced with IPTG and proteins separated by SDS-PAGE.
Results:
Genomic clones: 0/10 gave expression
cDNA clones: 2/10 gave expression
Question:
Why zero genomic clones
Why only 2 cDNA clones

2. Transgenic Organisms Lecture 6 Reading: Chapter 9
Molecular Biology syllabus web site

3. Genetic Markers RFLP/ RAPDS and other newer PCR-based methods
-to create maps
-to study evolutionary relationships
Mapping markers
-in situ hybridization, fluorescent tags
-Southern analysis (linked markers co-segregate)
-chromosome walking to generate physical maps
-comparison of physical and genetic maps

4. DNA polymorphisms can be used to map human mutations
Analysis of restriction fragment length polymorhpisms (RFLPs)

5. Isolation of a contiguous stretch of DNA and construction of a physical map in that region
Chromosome walking

6. Physical maps of entire chromosomes can be constructed by screening YAC clones for sequence-tagged sites
Ordering of contiguous overlapping YAC clones

7. Gene replacement and transgenic organisms
Some genes are identified through means other than mutant analysis
To determine the function of these genes, it is possible to replace an organism’s wild type gene with an inactive gene to create a “gene knockout”
It is also possible to introduce additional genes (transgenes) to create a transgenic organism

8. In vitro mutagenesis of a cloned gene
Gene knockout and
transgenic techniques
usually involve mutagenesis
of cloned genes prior to
transfer into the organism

9. Transgenic Approaches
Methods
spheroplasts-yeast, plants
chemical methods; microinjection- animal cells
electroporation
particle gun bombardment
bacterial-plants
Stable or transient
selection with markers
Knockouts (homologous recombination) “gene replacement”
Transgenic Organisms

10. Purposes of transgenic research
Basic- understanding gene function
Applied-
gene therapy to introduce functional genes
improvement (foods; create novel sources of drugs; increasing plant production to provide more food)

11. Creation of mice ES cells carrying a knockout mutation

18. Production of transgenic Drosophila
Eye color, a screenable phenotype encoded by w+ gene. Drosophila, red-eyed wild type (left) & white-eyed mutant (right).

19. Transgenic Plants
Plants cells are totipotent and can regenerate from undifferentiated tissue to produce viable, seed-bearing plants.
Methods:
electroporation, microinjection, bombardment, use of Agrobacterium tumefaciens

20. Production of transgenic plants with Ti plasmids

21. Reporter Genes as Transgenes
Examples
Advantage:
Easy to assay compared to native gene
GUS- b-glucuronidase
GFP- green fluorescent protein
LACZ- b-galactosidase
LUC- luciferase

22. Gene X is an enzyme,GGPPS
How do we determine where in the plant this gene is expressed?
Fuse the promoter of Gene X to the coding region encoding GUS (a bacterial enzyme, betaglucuronidase).
Assay enzyme activity of GUS using a chromogenic substrate. Active enzyme catalyzes formation of a blue product.

23. Reporter Genes as Transgenes Example: assaying the promoter of Gene X
Coding Region
ORF
Promoter
REPORTER
ORF

24. Reporter Genes as Transgenes
GUS
b–glucuronidase is a bacterial enzyme that acts on a chromogenic substrate to produce a blue product.
Arabidopsis promoter-GUS fusions expressed in Arabidopsis. (Okada et al., 2000, Plant Physiology 122: )

25. Artificial Promoters Promoter Coding Region Promoter Coding Region ORF
To alter natural expression with respect to time, place, or level of expression
Promoter
Coding Region
ORF
Promoter
Coding Region
ORF

26. Combining artificial promoters and reporter genes
Promoter for constitutive expression (35S)
GFP coding region
35 S Promoter
REPORTER (GFP)
ORF +
35 S Promoter
REPORTER (GFP)
ORF

27. Constitutive expression of GFP
GFP, Green Fluorescent Protein-
is a bacterial protein that will normally localize to the cytoplasm.
Transient expression of GFP in tobacco (Zhu, Li, Wurtzel, unpub.)

28. Gene X is a chloroplast protein
How do we determine which part of the protein is needed to direct it to a chloroplast
Fuse DNA encoding the putative transit sequence to the coding sequence of GFP (jellyfish green fluorescent protein) which is driven by a constitutive promoter (35S).
Use a fluorescence microscope to detect the fluorescence of GFP.

29. Promoter Coding Region 35 S Promoter REPORTER (GFP)
Combining reporters & constitutive promoters to assay gene elements Example: assaying transit sequence of Gene X
Gene X
Promoter
Coding Region
ORF
35 S Promoter
REPORTER (GFP)
ORF

30. Fusion of maize PSY transit sequence to GFP directs GFP to tobacco chloroplasts.
Untransformed PSY-GFP
Green
Red
Merged
Zhu, Li, & Wurtzel unpublished

31. Reporter Genes as Transgenes
GUS- b-glucuronidase
GFP- green fluorescent protein
LACZ- b-galactosidase
LUC- luciferase
Arabidopsis promoter-GUS fusions expressed in Arabidopsis. (Okada et al., 2000, Plant Physiology 122: )
Transient expression of GFP in tobacco (Zhu, Li, Wurtzel, unpub.)

32. Turning off genes Antisense Promoter Coding Region Promoter
ORF
Promoter
Coding Region
ORF

33. Turning off genes
RNAi