Class 11

1
Class 11

• Mouse technology continued
• Quiz 3 (classes 7-10)

2
We have discussed A few details about how
transgenic or chimeric mice are generated The
differences between transgenic and chimeric mice
How and why one might make these kinds of mice
We have NOT yet explained how you can
generate -knockout mice -targeted knockouts
(cre-lox)
Chimeric mice
3
How do you get ES cells that have undergone
homologous recombination?

• Constructs normally insert randomly into
  locations that have nicked DNA
• To get a homologous recombination, you have to
  select for cells that have swapped out their wild
  type copy of a gene for your mutated copy

4
Making knock-out mice using ES cells Technique
pioneered by Mario Capecchi See this website
with animations about the technique http//www.dn
aftb.org/dnaftb/41/concept/
G 4.21
You need a selectable marker Neomycin resistance
is part of the mutated piece of DNA If ES cells
are neo resistant, they contain the mutated piece
5
In fact, you need two selectable markers

• To select for a construct that has actually
  inserted into a specific location, have to use
  some tricks
• Positive selection neomycin resistance
  construct contains neo resistance gene any cell
  that has incorporated the construct (even
  randomly) will be resistant to neomycin
• Negative selection thymidine kinase (TK) from
  the herpes virus. If a cell contains the TK
  gene, it will be killed by the drug gancylovir

6
Remember, the ES cells are from a black mouse The
black coat color allele is dominant
G 4.21
7

• You transfect ES cells from a black mouse
  (dominant marker) with
• this construct
• Following transfection, the culture will include
• cells that have lost the transgene without
  inserting it
• cells that have inserted the transgene by
  homologous
• recombination at the YFG locus
• cells that have inserted the transgene at another
  locus by
• end-joining to a nicked strand followed by DNA
  repair.
• 1) In non-selective growth medium (no neomycin or
  gancyclovir)
• the most frequent class of cells will be ___.

TK
YFG
YFG
2) In medium containing gancyclovir alone, the
most frequent class of cells will be ___.
8
TK
YFG
YFG

• cells that have lost the transgene without
  inserting it
• cells that have inserted the transgene by
  homologous
• recombination at the YFG locus
• cells that have inserted the transgene at another
  locus by
• end-joining to a nicked strand followed by DNA
  repair.

3) In medium containing neomycin alone, the most
frequent class of cells will be ___ .
4) In medium containing neomycin and gancyclovir,
the most frequent class will be ___.
9
5. You inject embryonic stem cells that have
undergone homologous recombination at the YFG
locus into blastocysts obtained from a mated
white mouse and reimplant them into
pseudopregnant white females. When she gives
birth to offspring (Po mice) a) all will be
variegated (both black and white patches of coat
color). b) a few may be white, but the majority
will be variegated. c) some will be white, some
will be variegated.
10

• 6. You mate a variegated (chimeric) female mouse
  to a white male, and examine their offspring (F1)
  for coat color and the presence of the knocked
  out YFG. Which of the following statements is
  true?
• (Remember the white mice are homozygous black
  /black black coat color is dominant, and the
  gene is on a different chromosome from YFG).
• a) All of the black offspring are homozygous for
  the transgene
• (YFG-/YFG-)
• b) Some of the black offspring are homozygous
  for the transgene (YFG-/YFG-)
• c) All of the black offspring are heterozygous
  for the transgene
• (YFG-/YFG)
• d) All of the black offspring are homozygous
  normal
• (YFG/YFG)
• e) Some of the black offspring are heterozygous
  for the transgene (YFG-/YFG)

11
What if a null mutation is embryonic or
perinatally lethal? How could you determine the
function of the gene in the adult? How could
you determine the function of the gene in a
subset of cells?
It is possible to do this in the mouse using
Cre/lox technology
12
Tissue Specific Knockouts generate a transgenic
mouse where the gene of interest is knocked out
only in specific tissues Cre-lox mice

This is one mouseit carries the loxP sites
surrounding the piece of DNA you plan to remove
13

• Now, you generate a second mouse
• This mouse is transgenic for the Cre recombinase,
  driven by whatever promoter you choose

Mouse 2
Mouse 1
14

• Mouse 2 is transgenic for the Cre recombinase,
  driven by whatever promoter you choose

Mouse 2
Mouse 1
Now, mate the mice together
15
In cells where Cre protein is generated, it
induces recombination between the loxP sites,
removing whatever piece of DNA they were flanking
16
Cre-lox Strategy for Tissue-Specific Knockouts
Green regions BDNF removed by cre-mediated excisi
on of the loxP sites
Combine an allele having lox sites targeted to a
gene of interest with a tissue-specific Cre
transgene- this gives a tissue-specific (or
conditional) knockout
This should remind you of the GAL4-UAS system in
Drosophila (in that it is a targeted approach)