Mice and obesity

Question 1

What are the three genes in mice linked to obesity?

Answer 1

Obese, diabetic and non-agouti

Question 2

In the parabiosis experiment what happened?

Answer 2

When ob mice were joined to wild type, ob lost weight
When db mice were joined to wild type, db stayed fat and wild type lost weight (to the point of starvation if left for more than two weeks)
When db mice were joined to ob mice, the db mice stayed fat and the ob mice starved.

Question 3

What is the lipostat model?

Answer 3

Fat reserves in the body secrete a circulating factor which acts on a centre in the brain. High concentrations = loss of appetite and increased metabolism. Low concentrations = increased appetite and decreased metabolism.

Question 4

How was ob cloned?

Answer 4

Positional cloning

Question 5

How was db cloned?

Answer 5

candidate gene approach

Question 6

Where is ob located?

Answer 6

On chromosome 6; it had already been mapped between microphthalmia and waved-1

Question 7

What type of cross was performed for cloning ob?

Answer 7

an interspecific test cross, between Mus musculus and Mus spretus. Have highly divergent DNA at sequence level, and carry polymorphic forms of microsatellite and other DNA markers. Can interbreed; female F1 hybrids are fertile, backcrossed to male ob/ob mice producing an F2

Question 8

How many F2 progeny were produced in cloning ob and which were the tightest loci?

Answer 8

1602 F2 progeny produced; scored for ob phenotype and DNA extracted to score RFLP and microsatellite markers. Ob was most tighly linked to an RFLP in the PAX4 gene and D6Rck39; only 3/1603 showed recombination between ob and these marker loci

Question 9

What were the PAX4 and D6Rck39 markers used for?

Answer 9

To isolate 80-100kb clones from which to clone transcribed sequences in the interval between Pax4 snf D6Rck39 by chromosome walking.

Question 10

What is the molecular weight of leptin?

Answer 10

16kD

Question 11

Which chromosome was db mapped to?

Answer 11

Chromosome four, as well as the gene for the receptor isolated from foetal mouse brains which had a high affinity for leptin

Question 12

What are the mutational properties of db?

Answer 12

Db is abnormally spliced in one mutant allele and another contains a 17 bp deletion resulting in a frame shift mutation.

Question 13

How was non-agouti cloned?

Answer 13

positional approach; facilitated by mice which had chromosomal inversion with break points in non-agouti at one end and limb deformity at the other. Positional cloning of limb deformity allowed non-agouti to be cloned by chromosome jumping. Probes for limb deformity were used to screen a genomic library generated from inversion strain DNA and clones extending into the non-agouti locus from the inverted ld segment were isolated.

Question 14

What occurred after the clones of non-agouti were isolated?

Answer 14

The candidate region adjacent to the non-agouti breakpoint was screened for cDNA sequences (highly conserved in other mammals). Found to be expressed in the skin

Question 15

What is the agouti signal protein?

Answer 15

One cDNA was found and mutated in all mutants after cloning non-agouti, coding for a 130 amino acid protein. In the Avy dominant mutant agouti signal is structurally normal but misexpressed; detected at high levels throughout the hair growht cycle and ectopically in other tissues; brain, liver, lung, spleen and kidney. In wild type, agouti signal protein is only expressed in the skin at specific stages of hair growth cycle.

Question 16

What is the melanocortin pathway in coat colour and weight regulation?

Answer 16

Black pigment is produced in response to melanocyte stimulating hormone (MSH) binding to receptor (MCR-1) on the melanocyte cell surface. The non-agouti locu controls a switch from black to yellow pigment production by inhibiting MSH binding and is normally only expressed for a short period during hair growth.

Question 17

What is the MCR receptor family?

Answer 17

A family of receptors related to MCR-1 which can all bind MSH; if one o0f these is normally invovled in suppressing appetite ubiquitous non-agouti expression may inhibit this function causing Avy mice to get fat. 
MCR-1 = epidermis
MCR-2 = adrenal cortex
MCR-3 = brain, placenta, gut
MCR-4 = brain
MCR-5 = ubiquitously expressed.

Question 18

Which MCR receptors were knocked out?

Answer 18

MCR-1, MCR-3 and MCR-4:
MCR-1 are yellow but not fat so MCR-1 is not involved in weight control
MCR-3 have normal appetites but increased metabolic efficiency
MCR-4 exhibit hyperphagia.
Avy mutants are fat because ectopic a expression in the brain inhibits signalling from bothe the MCR-3 and MCR-4 receptors.

Question 19

What does FTO overexpression cause?

Answer 19

hyperphagia, FTO KO are growth retarded with reduced adipose and lean tissue due to increased energy expenditure.