HC6 transgenesis

Question 1

Introduction of non-permeable substances (DNA/RNA) into human cells: methods (6)

Answer 1

microinjection
electroporation
liposomes
particle bombardment
chemical compounds facilitate uptake of DNA pinocytosis

Question 2

transgenic animals: how to make them?

Answer 2

injecting DNA into pronucleus of zygote.
or removing DNA/nucleus from recipient oocyte and transferring it with an embryonic/somatic cell nucleus

Question 3

general vector

Answer 3

used for introduction of a gene.
needs:
- gene of interest
- selection marker

Question 4

knock-out vector needs:

Answer 4

• homologous DNA sequences
• positive selection marker
• negative selection marker: if it is expressed it indicates that the vector integrated randomly instead of via homolgous recombination. For instance tk1, tk2 genes

Question 5

zinc finger nucleases

Answer 5

artificial restriction enzymes that have a zin finger DNA-binding domain and a DNA-cleavage domain.
function as dimers: so pairs are required to target sequences

Question 6

TALEN nucleases

Answer 6

uses DNA binding motifs to direct a non-specific nuclease to the site of interest. Each domain recognises a single nucleotide

Question 7

CRISPR/cas9

Answer 7

a guide RNA seq is designed that is homologous to target sequence –> guide RNA binds Cas9 protein –> guides it to correct sequence –> protein cuts DNA.
Attempted repair will result in gene silencing or a repair template with a different sequence can be added –> gene will have a mutation (KO)

Question 8

AAV vectors

Answer 8

do not integrate in host genome, but genome persists in the nucleus as episomes, resulting in long-term transient expression of transgene.