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 transgene expression could be altered by its integration site in the genome?

1. Could integrate into heterochromatin (silent region) and not be expressed. 2. Could integrate next to the regulatory region of another gene and be expressed in a cell type that normally does not express it.

In gene targeting for a knockout of the Indy gene, what is the purpose of the positive selection cassette?

It selects for cells by providing resistance to a drug, ensuring only those cells with the integrated vector survive.

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

ES cells are pluripotent and can develop into any cell type, allowing changes to be passed on.

What are the two methods of DNA integration into the genome after electroporation?

Random transgenesis or homologous recombination.

Which method will not result in the successful knockout of the Indy gene?

Random transgenesis.

What is the next step after finding an ES cell colony with the Indy gene knocked out?

Inject ES cells into host blastocysts and transplant them into pseudopregnant female mice to make a chimera.

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

When Cre is expressed.

What type of protein is Cre?

A recombinase that recombines loxP sites and deletes the intervening sequence.

What are loxP sites?

Sequences that Cre recombinase binds to and recombines.

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 making it inosine, 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