Reverse Genetics and

Gene Knockouts

This meeting is being

recorded.

Molecular and Cell Biology

I BIOL-UA 21
Meeting 10
12 October 2021
Connecting genes and functions

“classical” or
“forward” genetics
Figure 6.1
Molecular Cell Biology, Eighth Edition
“reverse” genetics
© 2016 W.H. Freeman and Company

2
Knocking out a gene by homologous recombination

Figure 6.37b
Molecular Cell Biology, Ninth Edition
© 2021 W.H. Freeman and Company 3
2:0 segregation in progeny of a heterozygous diploid

4
What
What isiswrong
wrongwith
withthe mutants? (cell morphology?)
the
mutants?

Levy & Siegal, PLOS Biology 6:e264 5

What is wrong with the mutants? (growth rate?)

Matiza Sacotingo & Diego Quintana 6

Embryonic stem cells can be genetically engineered

Figure 22-4a
Molecular Cell Biology, Ninth Edition
© 2021 W.H. Freeman and Company 7
Gene targeting in ES cells

Figure 6.37a
Molecular Cell Biology, Eighth Edition
© 2016 W.H. Freeman and Company 8
Making a knockout mouse: inject ES cells into embryo

Figure 6.38
Molecular Cell Biology, Eighth Edition
© 2016 W.H. Freeman and Company 9
1
0
Injecting ES cells into an embryo

1
Practice question 1

You are knocking out the yeast LYS2 gene, which is

required for production of the amino acid lysine. Yeast
cells without the LYS2 gene will not grow unless their
medium is supplemented with lysine.
1) Draw a schematic diagram of the knockout construct you

would use to knock out LYS2 and replace it with the

selectable marker kanMX, which confers resistance to
G418.
2) Describe the procedure for knocking out LYS2 with this

construct and selecting mutant cells.

3) Describe how you could validate that the selected cells

indeed contain the desired knockout.

1
Practice question 2

1
Need for conditional disruption of gene
function What if a gene is required for early
embryogenesis and for ear development?

1
Two common methods of conditional disruption

knockout a gene during

development so it is
missing only in some
tissues

interfere with mRNA or its

translation only in some
tissues

1
Cre recombinase catalyzes site-specific recombination

white +
no Cre present

Cre present (in some cells)

white +

(lost during
cell division)
16
Making a conditional knockout mouse with Cre/loxP

Figure 6.39
Molecular Cell Biology, Eighth Edition
© 2016 W.H. Freeman and Company 1
Highlight: BRCA1 conditional knockout
mouse
BRCA1 knockout is recessive embryonic lethal
mammary-specific
BRCA1 promoter

What does this result say about tumor formation?

What would the consequence be of replacing the
mammary- specific promoter with a skin-specific
promoter?
What would the consequence be of replacing the
mammary- specific promoter with a promoter that is only
expressed in all cells of very early embryos?

Trends in Genetics 17:S18 1

Lowlight: Transgenic humans

Second International Summit on Human Genome Editing (November 2018) 1