Genetic manipulation technologies 2

Question 1

In what ways can we cause random mutagenesis?

Answer 1

Radiation

Chemical mutagens such as:

• ENU (ethylnitrosurea)
• EMS (ethyl methanesulphonate)

Question 2

How do ENU and EMS work?

Answer 2

ENU creates point mutations by ethylating DNA base pairs during DNA replication in sperm cells

EMS most converts G/C pairs to A/T pairs during DNA replication

Question 3

What technique is most commonly (at least in aberdream) for knocking out specific genes?

Answer 3

Gene knockout by homologous recombination in Embryonic stem cells

Question 4

What is gene knockout by homologous recombination? (briefly)

Answer 4

Very simplified but…

Gene target vector (transgene) introduced to the nucleus of ES cells. This gene is not functional but contains various markers

Homologous recombination happens when flank sequences of DNA on the host DNA and transgene line up

If they line up, the transgene is swapped in

Question 5

In the context of homologous recombination for gene knockout - what is positive and negative selection?

Answer 5

This gets rid of the unedited/incorrectly edited (ligated) chromosomes in the ES cells. Example…

When we carry out the process to cause homol. recomb of ES cells - most of the time, other integration events happen. Usually, the transgene is just attached to the chromosome randomly (thus has not knocked out the target gene)

Positive/negative selection aims to remove the 1) Unedited sequences and 2) the randomly integrated (ligated) sequences

Question 6

What is a gene knock in?

Answer 6

Same as homologous recombination gene knockouts except the inserted gene is actually functional

Question 7

Explain how positive/negative selection would occur for this…

• Transgene contains flank sequences
• Between flank sequences is a Neomycin resistance gene
• Outwith the flank sequences are Herpes simplex TK genes

Answer 7

After the process - you would expose the ES cells to:

1. Ganciclovir (antiviral) - cells with randomly integrated (ligated) sequences will still possess the Herpes simplex TK thus will die
2. Neomycin - cells that are unedited (ie do not contain the Neo. resistance gene) will die

The cells that are left must not have the TK and must have the neomycin resistance gene - ie must have undergone homologous recombination