MCB141 103/104
Quiz #5
1a. What are three features you should include when designing a gene-targeting vector for the
generation of mutant mice? Briefly describe the purpose of including each feature (1-2 sentences
per feature). 2 pts
- Flanking homology: direct the transgene to the desired site of integration by allowing for
homologous recombination
- A gene for positive selection (e.g., neomycin resistance): allow for the survival of cells that
contain the transgene.
Note that this may include “false positive” cells, i.e. cells in which random
integration, rather than homologous recombination, has occurred.
- A gene for negative selection (e.g., HSV-tk or another gene encoding a cytotoxic factor):
this will weed out random integration events
1b. Describe an assay you might use to verify proper vector integration, and the expected
experimental outcome (i.e. what you expect to see) if proper integration indeed occurred. 1pt
PCR, with one primer in target gene and the other primer in the transgene/insert.
Restriction enzyme digest using an enzyme that cuts in the transgene/insert sequence.
Sequence using primers that would generate a product including both endogenous and
transgene sequence
Or, sequence genome.
2. How do cells of the ICM differentiate from outer trophoblast cells? To what tissues will each
group of cells eventually contribute? 2pts
The mouse embryo is derived from daughter cells of the ICM, while extraembryonic tissues
are derived from daughter cells of the trophoblast. Trophoblast cells differentiate from
ICM cells at the 1632 cell stage transition. At this stage, the “outer” cells of the early
embryo upregulate the expression of Cdx2 and Gata3. These transcription factors
antagonize the expression of ICM transcription factors, thereby securing the cell fate of the
trophoblast cells.
3. How does the visceral endoderm contribute to the establishment of an anterior-posterior axis
in the mouse? 2pts
The cells of the distal visceral endoderm migrate anteriorly while secreting Nodal
antagonists Lefty1 and Cerberus. As the cells of the AVE continue to express Lefty1 and
Cerberus, Nodal signal transduction is downregulated in the epiblast neighboring the AVE.
Thus the anterior epiblast cells take on a distinct molecular identity from more posterior
epiblast cells.
4. How does the extraembryonic ectoderm contribute to the establishment of an anteriorposterior axis in the mouse? 1pt
The extraembryonic ectoderm secretes proprotein convertases, including Furin, which
process Nodal ligands. The faster processing of Nodal ligands allows for an initial spike in
Nodal signal transduction in the posterior, proximal epiblast.
In addition, BMP signaling in the extraembruonic ectoderm further amplifies Nodal
expression in the posterior, proximal epiblast.
Together, proprotein convertases and BMP signaling in the extraembryonic ectoderm
allow for high Nodal signaling in the posterior epiblast.
5. How does timing of the maternal to zygotic transition differ between frog and mouse
development? 1pt
MZ switch occurs at the 2-cell stage in mouse, but at the mid-blastula stage in frog.
Compared to frog, mouse oocyte cytoplasm doesn’t contain near enough RNAs and
proteins for cleavage and development
6. “Name that phenotype:” Based on what we’ve talked about in class, write in an “X” to
indicate the correct answer for each manipulation. VE= visceral endoderm
2pts
Less mesoderm
More mesoderm
No change
than usual
than usual
Knock out Lefty1
X
throughout VE
Nodal
uninhibited
everywhere
Mis-express Lefty2
X
throughout VE
Even though
not normally in
VE, still inhibits
Nodal
Knock out Nodal
X
throughout VE
Only needed in the
epiblast
Express FoxH1
X
throughout VE
Won’t affect
transcription
without Nodal
signaling
Knock out Furin in
X
extraembryonic ectoderm
Nodal ligands
will not be
processed as
quickly, lower
levels of Nodal
signaling