Lecture 1 Chromosome Basics

What part of a transgene controls which type of cell it will be expressed in?

• The promoter → housekeeping vs specific

What are two ways in which a transgene’s expression could be altered by the site in the genome that it integrated into?

You decide to study the function of gene called Indy which has 3 exons. You want to make a targeted knockout of this gene in mice, by inserting a resistance gene into the middle of exon 2, disrupting it.

1. Draw the structure of the targeting vector you would need to make. Label the parts of your vector. What is the purpose of each part?

   • see slides

   • 5’ - positive selection - 3’ - negative selection

   • arms provide homology so recombination will occur

   • positive selection cassette selects for cells by providing resistance to a drug that would kill the cells if the vector is not integrated

   • negative selection cassette kills cells where entire vector integrates and there wasn’t homologous recomb. just random insertion

2. Which part(s) of your vector contain Indy gene?

   • the 5’ and 3’ arm

3. Why does gene targeting have to be done in ES cells?

   • ES cells are pluripotent, can develop into any cell types and changes can be passed on

4. Now that you've electroporated this vector into the ES cells, what are two ways that your DNA can integrate into genome?

   • random transgenesis or homologous recombination

5. Which way will not result in the successful knockout of Indy?

   • random transgenesis

6. You've found an ES cell colony with the Indy gene knocked out. What is the next step?

   • inject ES cells into host blastocysts and transplant into pseudopregnant female mice → makes chimera

When does gene knockout occur in conditional mutagenesis Cre/LoxP systems?

• when Cre is expressed

What kind of protein is Cre?

• a recombinase - recombines loxP sites and deletes intervening sequence

What are the loxP sites?

• sequences that Cre recombinase binds to and recombines

Redraw the vector from question 3a, but this time make it conditional. Put loxP sites on either side of exon 2.

• see slides

• lox P sites around exon you want to knockout

What are the three essential elements of the ABE base editing system and what is the role of each?

• nucleoside deaminase - removes an amino group from adenine (A) making it inosine (I) which the cell interprets as a G

• guide RNA - complementary to sequence in genome where editing takes place

• nCas9 - nicks the strand bound by gRNA, encourages cell to use inosine as template for repair of G-T mismatch