Next lecture:techniques used to study the role of genes in develpoment

• Random genetics followed by screening

• Targeted mutagenesis (gene knockout)

• Transgenic animal models

• Dominant negative mutant molecules

• Antisense RNA interference

• RNA interference (c. elegans-website 4.8)

Random genetics

• Chemical mutagen/gene disrupting agent

• Screening for a phenotype

• Dominant mutations can be found easily

• Recessive mutations require breeding

• Frequently used with model organisms– Especially Drosophila (lectures in late Feb.)

• Becoming newly popular in the mouse

Mouse ENU mutagenesis

Recent applications of this technology can be seen in: Nature Genetics,(Aug 2000)Volume 25 pp.440-443 and 444-447

Trans-heterozygous phenotypes

• When two genes are in the same “pathway” mutants heterozygous for both genes will display a phenotype even though each individual heterozygous mutant does not

• Can be combined with ENU mutagenesis to screen for genes in the same pathway as another known “knocked out” gene.

Targeted mutagenesis (knockout)• Determine the action of a known, cloned

gene in a developmental process

• Removes a segment of the known gene by homologous recombination

• Required elements:– Mapped genomic clone for the gene of interest– Embryonic stem (ES) cells– A lot of repetitive work

Points to make

• Genomic clone should come from the same mouse strain from which the ES cell is derived (common strain 129SV)

• Making of targeting construct. 6kb of homologous arms with appropriately arranged selectable markers for positive and negative selection

• A screening strategy involving two probes

X N B B E B X

10 kb2 kb

X N B B E B X

10 kb2 kb

5’ arm 3’ arm

X N B B E B X

10 kb2 kb

5’ arm 3’ arm

ABC loxP neoR loxP

DEFTKS

X

X N B B E B X

10 kb2 kb

5’ arm 3’ arm

loxP neoR loxP TKS

X

Targeting vectorN

X N B B E B X

10 kb2 kb

5’ arm 3’ arm

TKS

X

Targeting vector

N

X N B B E B X

10 kb2 kb

5’ arm 3’ arm

TKS

X

Targeting vector

N

X N E B X

10 kb2 kb

X

Homologous recombinant

X

Targeting vectorRandomly integrated

X N E B X

10 kb2 kb

X

Homologous recombinant

X

Targeting vectorRandomly integrated

TKS (gancyclovir)

X N E B X

10 kb2 kb

X

Homologous recombinant

X

Targeting vectorRandomly integrated

5’ probe 3’ probe

Note, probes are OUTSIDE the homology arms. Therefore, they will only detect the endogenous locus and the recombinant

Removing the selection marker• Cre recombinase deletes sequences between

two lox-P sites in the same orientation

• Transiently transfect a vector expressing the cre recombinase for deletion

• Grow clones again and screen for the deletion by southern blot as before

• Especially necessary in studying knockouts of genetic regulatory sequences

Cre-mediated deletion

X N E B X

10 kb2 kb

X

Homologous recombinant5’ probe 3’ probe

Cre expression vector

X N E B X

Cre-deleted recombinant5’ probe 3’ probe

The next steps

• Grow up clones with correct recombinant

• Inject ES cells into mouse blastocyst (d3.5)

• The ES cells will integrate themselves into the blastocyst and mouse will be a chimera

• Usually the ES cell strain has a different coat color than the blastocyst strain so that the “marbleized” mice can be easily seen

And then….See also fig 4.19 on page 98 of Gilbert

• Breed the chimeric mice to normal mice

• If the ES cells contributed to the germline the babies which contain the mutation will have the coat color of the ES cell– These mice are only HETEROZYGOUS for

the targeted gene

• These mice need to be bred to homozygous

• Analyze the phenotype…...

Further references on homologous recombination in ES cells

• Capecchi, MR (1989) Altering the genome by homologous recombination. Science. 244:1288-1292.

• Ramirez-Solis, R, Davis, AC and Bradley, A. (1993) Gene targeting in embryonic stem cells. Meth. Enzymol. 225:855-875.

Possible phenotypes

• Something related to what you expected

• Something completely unexpected– No phenotype– Embryonic lethal– Complex phenotype-multiple tissues and

effects

Conditional gene targeting

• Tissue-specific knockout of a gene– Avoids embryo lethality– Avoids complex phenotypes

• Inducible knockout– Allows “before” and “after” type analysis– Model of acquired mutation rather than

inherited mutation

Strategy (from Rajewsky, et. al.)

Transgenic animals (mice)

Points to make

• Transgene should be free of vector DNA

• Transgene must be rigorously purified

• Transgene integration is a bit inefficient

• Founder mice are sometimes mosaic

• Need to outcross the mice to wild type mice

• The transgene is not always expressed

• Position-effect-variegation

Transgenesis and developmental studies

• Inappropriate or overexpression of a gene

• Dominant negative mutant gene expression

• Reporter gene expression for lineage tracing– Fluorescent proteins (GFP, YFP)– Beta-galactosidase (X-gal staining)

• Transcriptional regulatory elements– Assuming a position-independent system

Antisense and RNA interference

• Overexpression of anti-sense RNA– Not the method of choice though it has worked

in some instances.

• RNA interference in c. elegans– Website 4.8

Model of RNA interference

RNA interference in C. elegans

Next lectures: Differential Gene expression

• Chapter 5 and websites on syllabus

• Epigenetic control mechanisms– Histone modification– DNA methylation– Nucleosome disruption “machines”

• Promoters and enhancers– Old and new models of enhancer function

• Novel transcriptional control sequences