Transgenesis, preimplantation, stem cells, and chimeras Flashcards
Tutorial 1 Qs and Answers
You identify a mouse mutation which interferes with the formation of the lungs during development. At what stage of development do you expect such mutant embryos to die and why?
Only postnatally and not as embryos, since the lungs are not required for embryonic development. They are required immediately after birth, so their absence would cause an early postnatal lethality.
Several mouse mutants die around mid-gestation (E10). What are the main possible reasons for embryonic lethality at this developmental stage?
Vascular defects, implantation/placental defects, heart defects
What are the polar bodies? How could you test the developmental potential of the polar bodies? Which of the 2 polar bodies would you use for your experiment and why?
They are the products of meiosis in the female germline, discarded from the egg. The nucleus in the polar bodies could be injected in an enucleated egg, which could than be fertilized. This would allow to test whether the chromosomes in the polar bodies can support normal development. The second polar body should be used, since it is haploid, as the normal maternal pronucleus.
Using fancy new technologies, you disaggregate all the cells of a late blastocyst and determine which genes are expressed in each individual cells obtained. You find that based on these so-called single-cell expression profiles, all the cells can be grouped into 4 distinct categories. Name the 4 cell types of the blastocyst which these categories might represent. Which one(s) express Cdx2? Oct4? Gata6?
Epiblast (Oct4+), primitive endoderm (Gata6+), polar and mural trophectoderm (both Cdx2+)
You identify a new gene expressed during preimplantation development, Your favourite gene (Yfg). Using quantitative RT-PCR, you measure the level of Yfg mRNA in zygotes and 2-cell stage embryos and find that the levels go down between the 1- and 2-cell stages. What can you conclude from this observation? And what if the levels at the 4-cell stage are higher than those in the zygotes?
How could you independently confirm your interpretation of the latter experiment?
Yfg is a maternally deposited mRNA, already present in the egg. It starts to be degraded at the 2-cell stage but after ZGA it accumulates to higher levels. Look for expression of a paternal variant of Yfg: only the maternal copy should be detected at the 1- and 2-cell stage, but transcription at the 4-cell stage should include paternal mRNA as well.
You decide to study the function of Yfg in development and generate a null allele by gene targeting in male (XY) ES cells.
a) Draw the structure of a simple targeting vector to generate a knock out allele of this gene in embryonic stem cells
b) Identify and explain the purpose of the key parts of your vector.
a) Arms of homology flanking the region to be deleted, for homologous recombination - a positive selectable marker, such as neo, in between the arm, to select for insertion of the transgene - a negative selectable marker, outside the arms, to enrich for homologous recombination
c) Upon introduction of your linear DNA construct in ES cells, explain two different mechanisms for its integration.
Random or targeted insertion.
d) What component of the targeting vector can enrich for what you want to obtain?
The negative selectable marker, by eliminating clones in which the construct integrated randomly, like a transgene. If targeting by homologous recombination occurs, this marker will be excluded from the inserted DNA.
e) So now you have heterozygous Yfg+/– ES cells. Explain all the steps you will have to perform to go from these ES cells to your first Yfg homozygous mutant mouse embryo for phenotypic analysis.
make chimeras with the heterozygous ES cells, via injection or aggregation chimeras with an albino host embryo - obtain chimeric males, based on their coat colour, and mate them with albino females. This way, only transmission of the ES cell genome will give pigmented progeny. These have 50% chances of carrying the mutation: these are the heterozygous founders of your colony. - cross heterozygous male and female mice - 25% of their progeny will be homozygous for the mutation and can be analysed for an abnormal phenotype.
What is the difference between mosaic and chimeric?
Mosaic: single zygotic origin (2 parents) but genetic heterogeneity in somatic cells.
Chimeric: at least 2 zygotic origin (4 parents). It is the random mixing of cells of originating from two separate, different embryos.
What do we mean by germline transmission?
It is the fact that ES cells in a chimera manage to go through the germ line, to make a mature gamete, which can be passed on to the progeny. For this to happen, the starting ES cells need to have a good (pluripotent) developmental potential and contribute to the germline. Such “good” ES cells are said to be “germline-competent”.`
How do we screen for germline transmission from a chimeric male?
Cross a chimeric male, defined by the fact that its coat colour carry pigmented spots, to an albino female. Each time the ES cell genome makes it into a fertilizing sperm, the progeny will be pigmented, since this trait is dominant over albino.
Why do we breed chimeric males, not females?
The male, XY, ES cells are better ES cells, easier to work with, and these can differentiate into sperm if they contribute to the germline in a male. But more importantly, males can give so much more progeny than females, since they can be crossed to several different females each week. This allow for a much faster identification of germline transmission, since it’s essentially a numbers game. In some chimeras, ES cells contribute only to a small percentage of all the germ cells.
Where are red blood cells produced in the conceptus, first? And then?
The first site of hematopoiesis is the yolk sac, an extra-embryonic structure. Eventually this role is take over by the fetal liver.
What are loxP sites and what are they used for?
They are short recognition sites for the Cre recombinase. They allow to engineer alleles, called conditional mutations, that can be activated or mutated (deleted) only where and when the Cre protein is produced. The tissue-specific expression of Cre will determine where the mutation will be induced, by deleting the DNA sequences located in between the two loxP site. Thus, the mutation is conditional.
