Transgenics and Knockouts

Question 1

Transgenics

Answer 1

Extra genetic material is integrated into the genome

Introduce foreign DNA into early embryo to ensure all cells have the desired gene

Observe what happens to an entire organism during development when this DNA is expressed in cells

Transgenic mice have become models for studying human diseases and developmental defects.

Question 2

Creation of a Transgenic Mouse

Answer 2

Question 3

Transgenic Embryo

Answer 3

Gene of interest:
Hox gene

Observation:
Dark areas show presence of Hox gene expression

Interpretation:
High activity of Hox gene is present in the mesenchyme of the developing limbs and regions in the head.

Question 4

Nobel Prize for Transgenic Mice

Answer 4

In 2007, the Nobel Prize in Physiology and Medicine was awarded to Mario R. Capecchi, who shared it with Sir Martin Evans, and Oliver Smithies for their work on “principles for introducing specific gene modifications in mice by the use of embryonic stem cells”.

Question 5

Knockouts

Answer 5

Genetically engineered mouse in which both alleles of one gene are non-functional.

Observe what happens to a knock-out organism during development to learn about a gene’s function.

Knock-out mice have become models for studying human diseases and developmental defects.

Making mESC with one mutated copy of YFG

Select your gene of interest

Make targeting construct (TC) by inserting neomycin cassette (neocassette) into an exon to disrupt (mutate) the gene and add thymidine kinase to the end of the gene to later verify proper insertion by homologous recombination

Introduce the TC into the mouse embryonic stem cells (mESC) by electroporation

Place the mESC in medium with G418 (antibiotic) to select those with the TC

Only cells that have the TC with the neocassette will be able to grow.

Now you have cells with one copy of the TC obtained through positive selection

Question 6

Knock-Out MiceSelecting for homologous recombination

Answer 6

If TC is inserted by homologous recombination, TK will be lost.

If TC inserted randomly. TK will be retained.

Use negative selection to remove cells with TK

Presence of TK makes cells susceptible to ganciclovir

Cells that have undergone non homologous recombination and have the TK insert are not able to grow.

Cells that have the knock-out gene of interest can be removed through negative selection. TK removes random inserts

Question 7

Knock out mice final steps

Answer 7

Question 8

Knockout mouse summary

Answer 8

Question 9

Knock out mouse…does it work?

Answer 9

Question 10

Transgenics vs. Knockouts

Answer 10

Transgenic
Add a foreign gene
Examine effects due to gene expression
Reveals role of gene in development and disease

Knock-Out
Mutate an endogenous gene
Examine effects due to lack of functional gene
Reveals role of gene in development and disease