How do we Isolate Genes involved in Development?

Mutagens
Lecture# 7 BIOL 430 January 23rd 2006 How do we identify genes involved in development? Making mutants and transgenic animals

Type of Mutagen Type of DNA Damage

Chemical mutagenesis(i.e. EMS or ENU
N-ethylinitrosourea (ENU) ehtylmethanesulfonate (EMS)

Applications
Saturation mutagenesis, efficient (1/1000), random, isolate any type of mutation lof or gof not quite random, provides a tag for cloning, isolate loss of function mutations as well as gain of function mutations inefficient (1/10 000) random, usually isolate null mutations

Base Pair Change point mutation GC to AT

Transposons (i.e. P element DNA insertion or Tc1)

Irradiation (X rays rays)

Deletions and chromosomal rearrangements

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

Identification of recessive Mutations in zebra fish

Male fish are mutagenized (ENU)

Haploid embryos in zebra fish

UV
female derived from a mutagenized male cross

rare event m/+ leads to mutation

m/+ +/+

+/m

1000s of individual crosses required

or
+/+ m/+ +/+

or
m/+ Raise families 50% are +/+ 50% are =+/m m/+ Only the double het cross will give rise to 25% mutant progeny

m/+

+/+

+/+

m/+

+/+

+/+

m/+

Do the haploid embryos show phenotype?

25% of progeny mutant

Fig. 2.44

can maintain as homozygote or as a het if the mutation is lethal BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

Concept of a Balancer Chromosome

Balancer Chromosome inversion let

Isolation of recessive embryonic lethals on a certain chromosome

a a

b b

e e

f f

gfp

3 c d X
gene c is recessive lethal mutation

cross

X 5

let

a a

b b

e e

f f

gfp

3 c d X

29oC

Female genotype: DTS= dominant temp sensitive b= recessive lethal Male genotype: a= recessive marker ie. white (same chromosome asb mutation) 1. mutagenized males-potential mutation in sperm 2. Select for male cross progeny at 29C only a*/b (a*/DTS dead) use these males to cross to DTS/b females 3. Select at 29C only get a*/b cross progeny (males and females) If a recessive lethal linked to a then you will not see flies with white eyes -score dead embryos for pattern defects (non house keeping genes)

let let

a a a a a a

b b b b b b

c c

d d

e e e e e e

f f f f f f

gfp gfp

25% lethal homozygous balancer

let

c d c d X c d X c d X

gfp

50% viable heterozygous balanced stock (GFP positive) 25% lethal homozygous mutation

Properties of a Good Balancer Chromosome: 1. Should suppress recombination in the region (inversions) 2. Have a dominant phenotype (i.e.GFP) and recessive phenotype (lethal) 3. Must have a wt copy of the gene

no white eye Box 2B

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

Isolating Mutants in C. elegans

Mutagen Generation Physical: X-Ray Chemical:EMS (Ethyl Methane Sulfonate)

oocyte genotype sperm genotype wild type (+)

wild type (+)

mutation (mut)

Po

+/+
self fertilization

+/+
25%

+/mut
25%

F1

mut/+

+/+
F2

mut/+

mut/+

mut/mut
(25%)

+/mut
mutation (mut)
F3

mut/mut
25%

25%

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

What type of mutants can we isolate in C. elegans?

Morphological: ones such as bumps on the head and tail and along the body defects Examples: vab (variable abnormal) worms that lack vulvas, or have too many (vul) Longer thin worms (lon) Wild type Small or short and fat worms Dumpy (sma) (dpy)
vab-1 Behavioral: Uncoordinated movements (unc) (many cloned: axon guidance mutants and muscle defective)

How to generate Transgenic Animals? Mouse Fly Worm

Cell Lineage abnormal: (lin)

Embryonic Lethal mutants:(let) Sex Determination: Sexual transformation mutations: male transformer (tra) .
BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

Gene Targeting in Mouse

Why make transgenic animals?
Find out where a gene is expressed (i.e. reporter constructs) Over express or ectopically express a gene (i.e. create a dominant gain of function phenotype) Knock out a gene Genetic mosaic analysis Clone a gene by transformation rescue
BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

ES cells are derived from the ICM

By electroporation Or viral infection

See Box 4B and 3C

Continued

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

Gene Targeting in Mouse

ES cells are selected for targeted gene mutation

Microinjection in C. elegans
Inject DNA here
eg. GFP construct + injection marker (Roller)

Mix ES cells with a blastocyst from a host mouse

mitotic germ cells (syncytium)

Transplant blastocyst into uterus of host mother Donor ES cells from brown mouse host mother White mouse. Establish chimera lines and hope that some of The ES cells were incorporated into the germline

Box 3C

http://www.mcb.arizona.edu/wardlab/injectionvid.html
BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

GFP transgenic worm with Roller marker

Drosophila P-element mediated transformation

P-element: Transposons that insert almost any site on a chromosome in the germline and requires an enzyme called transposase. Two plasmids are injected into posterior end (pole cells) of fertilized eggs from white(-) parents. One plasmid has a defective P element such that it cannot hop and is also marked i.e. white (+) this plasmid carries your gene of interest. The other plasmid is a helper P-element that has transposase but cannot insert so it is easily lost. Embryos that survive the injection grow up as white eye flies and some may have a P-element inserted in a chromosome in the germline. Mating between these flies will lead to second generation with flies that have an integrated P-element and can be distinguished by red eyes.

The injected DNA gets into the nuclei and exists as an extrachromosomal array (multiple copies of the genes) and is lost randomly during mitosis. You can maintain the transgenic animals by picking the marker phenotype (i.e. Rollers) or integrate the DNA array into the chromosome by irradiation (i.e.X-rays)
BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

Transgenic fly Box 5A

Uses: 1)increase the copy number of genes 2) LacZ or GFP constructs 3) GAL-4 system

BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang

UAS GAL4 expression system.

GAL-4 Expressing Line UAS-target gene line

endogenous promoter/enhancer sequences (GAL-4 is expressed in a cell or tissue specific pattern)

GAL-4

X
UAS

target gene
(GAL-4 binding sites) Target gene is off in the absence of GAL-4

GAL-4 target gene GAL-4 drives UAS-target gene in a cell or tissue specific pattern
BIOL 430 2006 Molecular Genetics of Development Dr. Ian Chin-Sang