Transcript Slide 1

Problem
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
Lecture 6
Transgenic Organisms
Reading:
Chapter 9
Molecular Biology syllabus web site
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
DNA polymorphisms can be used to
map human mutations
Analysis of restriction fragment length polymorhpisms (RFLPs)
Isolation of a contiguous stretch of DNA and
construction of a physical map in that region
Chromosome walking
Physical maps of entire chromosomes can be
constructed by screening YAC clones for
sequence-tagged sites
Ordering of contiguous overlapping YAC clones
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
In vitro mutagenesis of a cloned gene
Gene knockout and
transgenic techniques
usually involve
mutagenesis
of cloned genes prior to
transfer into the
organism
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
Purposes of transgenic research
• Basic- understanding gene function
• Appliedgene therapy to introduce functional
genes
improvement (foods; create novel
sources of drugs; increasing plant
production to provide more food)
Creation of mice ES cells carrying a
knockout mutation
Production of
transgenic Drosophila
Eye color, a screenable phenotype
encoded by w+ gene. Drosophila, redeyed wild type (left) & white-eyed mutant
(right).
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
Production of transgenic plants with Ti plasmids
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
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.
Reporter Genes as Transgenes
Example: assaying the promoter of Gene X
Gene X
Promoter
Coding Region
ORF
Promoter
REPORTER
ORF
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 122:1045-56.)
Artificial Promoters
To alter natural expression with respect to time, place, or level of expression
Promoter
Coding Region
ORF
Promoter
Coding Region
ORF
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
Constitutive
expression of
GFP
GFP, Green
Fluorescent Proteinis a bacterial protein
that will normally
localize to the
cytoplasm.
Transient expression of GFP in tobacco (Zhu, Li,
Wurtzel, unpub.)
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.
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
Fusion of maize PSY transit sequence to
GFP directs GFP to tobacco chloroplasts.
Untransformed
PSY-GFP
Green
Red
Merged
Zhu, Li, & Wurtzel unpublished
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 122:1045-56.)
Transient expression of GFP in
tobacco (Zhu, Li, Wurtzel, unpub.)
Turning off genes • Antisense
Promoter
Coding Region
ORF
Promoter
Coding Region
ORF
Turning off genes
• RNAi