Problem

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