Lecture 7

Question 1

Lac operon (3 facts)

Answer 1

Suite of genes in bacteria that is only expressed when lactose is present and glucose is absent IPTG used in lab to induce Lac operon expression The promoter can be placed in any ol’ plasmid to express any gene in the presence of IPTG

Question 2

Lac operon in eukaryotic cells (e.g. yeast, Chinese hamster ovarian cells)

Answer 2

Lac operon is in a plasmid together with target gene and viral origin of replication Plasmid is introduced into cells through lipid treatment or electroporation. This is called transfection. Transfection can be transient (not every daughter cell has vector) To ensure stable transfection into genome, include resistance to G418 in plasmid, and then apply G418 to kill other cells

Question 3

Use of eukaryotic Lac operon (molecule, disease, drug name)

Answer 3

β-glucocerebrosidase, Gaucher disease, Cerezyme

Question 4

Yeast two-hybrid system (2 facts)

Answer 4

Bait plasmid and prey plasmid used When genes on both plasmids are expressed, protein products interact, and reporter gene is activated *If you can, look this up and know it better. It’s still a bit over my head.*

Question 5

Transgenesis

Answer 5

Method of in vivo characterization of gene/protein function Performed in fertilized egg before fusion of male and female pronuclei

Question 6

Green fluorescent protein (3 facts)

Answer 6

Used as reporter for gene or protein function Reports location of gene function when bound to promoter (promoter-fusion) Reports location of protein when expressed within gene of protein (protein-fusion)

Question 7

How to Make a Knockout Mouse: The Beginning

Answer 7

Begin with embryonic stem (ES) cells. They’re pluripotent. Cells integrate the target gene into genome using both homologous and non-homologous recombination. Both attempt to integrate the target gene, which codes for neomysin resistance (neo-) and thymidine kinase, into the genome. Homologous recombination integrates only neo- into genome; non-homologous recombination integrates the whole gene.

Question 8

How to Make a Knockout Mouse: The Selection

Answer 8

G418 kills cells that did not recombine at all. Ganciclovir kills cells that performed non-homologous recombination. This works because ganciclovir with thymidine kinase is toxic.

Question 9

How to Make a Knockout Mouse: The Birth

Answer 9

Recombinant ES cells combined with wild-type embryo

Chimeric pup results if the cells incorporated. Solid black pup results if not.

Continue mating until all offspring display knockout phenotype.

Question 10

Genome editing (3 types)

Answer 10

Zinc finger nuclease (ZFN) is in clinical trials

TAL effector nuclease (TALEN)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR!!!!!!!!!!), and CRISPR-associated (Cas) system

Question 11

Conditional knockout mice

Answer 11

Mice with genes that can be knocked out after exposed to external substrate. Useful for knocking out genes crucial for development.