problem
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
Problem1. You screen two libraries- cDNA; genomic2. Clones are isolated having homology to PSY- 10 clones
from each library3. 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 expressionQuestion:
Why zero genomic clonesWhy 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 sitesOrdering 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 andtransgenic techniques usually involve mutagenesisof cloned genes prior to transfer into the organism
Transgenic Approaches
• Methodsspheroplasts-yeast, plantschemical methods; microinjection- animal cellselectroporationparticle gun bombardmentbacterial-plants
• Stable or transient selection with markers• Knockouts (homologous recombination) “gene
replacement”• Transgenic Organisms
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)
Creation of mice ES cells carrying a knockout mutation
Production of transgenic Drosophila
Eye color, a screenable phenotype encoded by w+ gene. Drosophila, red-eyed 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
• GUS- -glucuronidase• GFP- green fluorescent
protein• LACZ- -galactosidase• LUC- luciferase
Examples
Advantage:
Easy to assay compared to native gene
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 TransgenesExample: assaying the promoter of Gene X
Promoter Coding Region
ORF
Gene X
Promoter REPORTER
ORF
Reporter Genes as Transgenes
GUS
–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 PromotersTo 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
GFPGFP, Green Fluorescent Protein-
is 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
Promoter Coding Region
ORF
Gene X
35 S Promoter REPORTER (GFP)
ORF
Untransformed PSY-GFP
Green
Red
Merged
Zhu, Li, & Wurtzel unpublished
Fusion of maize PSY transit sequence to GFP directs GFP to tobacco chloroplasts.
Reporter Genes as Transgenes
• GUS- -glucuronidase• GFP- green fluorescent
protein• LACZ- -galactosidase• LUC- luciferase
Transient expression of GFP in tobacco (Zhu, Li, Wurtzel, unpub.)
Arabidopsis promoter-GUS fusions expressed in Arabidopsis. (Okada et al., 2000, Plant Physiology 122:1045-56.)
Turning off genes • Antisense
Promoter Coding Region
ORF
Promoter Coding Region
ORF
Turning off genes • RNAi