Somatic cell cloning
Even though we are not in the BMS program, let’s talk a bit about the immune system. Our immune
system needs to defend us from a vast array of pathogens, and the first step to killing a pathogen is
identifying it.
For many ubiquitous pathogens, our innate immune system is able to recognize things that obviously
don’t belong. For example, cells of the innate immune system express receptors that recognize
lipopolysaccharide (LPS) found in the outer membrane of all Gram-negative bacteria. However,
pathogens are constantly evolving and their surface markers can adapt to evade detection. How can we
hope to recognize the vast number of ever-changing pathogens that want to infect us?
Solution: Randomly generate a ton of receptors that can detect all sorts of cell surface markers – both
host and pathogenic – then discard any of them that recognize host surface markers. Each antibody
requires a heavy-chain with a Variable (V), Diversity (D), and Joining (J) domain. The germline genome
has 51 V, 27 D, and 6 J regions which can generate 8262 heavy chain combinations. This number is
further inflated by pairing these heavy chains with over 200 possible light chain combinations, as well as
“junctional diversification” (random loss and addition of nucleotides at the splice site), to yield at least
1.6 million possible antibodies.
Mature B-cells, however, express only a single VDJ combination. They do this by somatic recombination
events that occur only in the bone marrow of a developing immune system. As VDJ regions are
randomly recombined together, the unused regions are flipped out of the genome and degraded.
http://laikaspoetnik.wordpress.com/2008/12/
In class on Monday, Nirao mentioned that complete mice had been cloned from individual somatic cells.
In broad terms, how could this type of experiment demonstrate that the genome of certain somatic cells
has undergone a permanent change?
If you clone an entire animal from a single somatic cell that has already undergone a permanent change,
then the entire animal generated should show exhibit the same change.
If a complete mouse were to be cloned from a single, mature B-cell, how would its genome differ from
the parent mouse? How could you observe this at the cellular level? What would you predict for the
immune health of the animal?
The VDJ region will be pre-recombined, and will not be able to generate the variety of antibodies that
the parent could.
Each B-cell should express the exact same surface antibody. Isolate B-cells and PCR over the V-D-J
region. You should observe a smaller band size if a region has been recombined out.
Severely immune compromised. Only able to detect pathogens that bind to one type of antibody.
This experiment hasn’t actually been done, but a using blood cancer cell line (clonal for the VDJ region
as cancer would develop from a single cell population) to generate a mouse embryo (dies before birth,
as it is a cancer cell line) can produce immune cell precursors that all have one type of VDJ region.
When it was discovered that each olfactory neuron expresses a single GPCR, it was hypothesized that
irreversible recombination events similar to those seen in B-cells could be the mechanism for selecting a
single receptor. How could you investigate this by cloning an entire animal from a single somatic cell?
What cellular changes would you look for in the cloned animal? What phenotypic changes?
Clone from a single olfactory neuron
If irreversible recombination has occurred – all olfactory neurons should express one GPCR. The mouse
should be sensitive to an extremely limited range of odors.
If regulation of GPCR selection is reversible – olfactory neurons should express the full array of GPCRs.
The mouse should be normally sensitive to odors.
Experimentally, mice cloned from olfactory neurons displayed a full array of GPCRs. However, Nirao also
mentioned that the mechanism for selecting a single receptor was known to depend on recombination
machinery. Propose a recombination-based mechanism by which a single receptor can be expressed,
while the entire array of receptors is maintained in the cells.
Think back to the yeast mating type locus. There is one transcriptionally active site, and the array of
receptors is maintained in a silent region. The one receptor to be expressed is recombined into the
active region.
Identifying stem cells and characterizing their dynamics
The intestine contains multiple different cell types with specialized roles in processes such as nutrient
absorption and mucus secretion. These cells turn over extremely rapidly implying that there must be a
stem cell population that replenishes the tissue.
For a long time the stem cells remained unidentified. Why might this be?
Ans: the def of a stem cell (self renewing and multipotent) is functional. There’s no inherent
morphology or molecular markers for a stem cell.
Baker et al discovered a gene LGR5 that, by in situ, is only expressed by a handful of cells at the base of
the intestinal crypts. These cells had no previously characterized function. How would you determine
whether they were stem cells? What mice would you use and what controls would you have to do?
You need to do fate mapping. They did w/ a knock in allele of LGR5-GFP-IRES-ERcre and Rosa26-loxPstop-loxP-lacZ. The main thing you have to worry about is whether LGR5 is really only expressed in
the cells at the base of the crypts since transient expression elsewhere will lead to permanent
marking of the cells. The GFP helps by letting you look to see that your not getting expression
elsewhere. Even more helpful is making the cre drug inducible since when you apply a pulse of
tamoxifen you’ll activate recombination only the cells expressing cre at that time which by and large
will be the cells you want. It was also very reassuring that they saw the predicted pattern of
outgrowth you would expect from a clone originating from crypt base cells.
You find that the clones you generate give rise to all cell types. After you break out the champaign and
submit to nature, the bioreg professor on your thesis committee asks if you’ve proven that all LGR5
intestinal cells are stem cells. What do you say?
No. You showed that there’s a stem cell pool within the LGR5 pos cells but its possible that LGR5
labels a heterogeneous population.
Making Technicolor mice!
Intro:
Mouse intestinal stem cells are found at the base of mouse intestinal crypts as pictured above. Figure E
shows a horizontal cross section of a mouse intestinal crypt. In these intestinal crypts stem cells can be
replaced. You are interested in studying the rate of stem cell replacement. In general, what would you
need to be able to study stem cell replacement?
In principle, you would need to label cells in different color and then measure the increase in clone sizes
of different colors over time
Luckily, someone has already invented a tool that will label tissues different colors by taking advantage
of the lox-cre system along with some cleverness involving gene inversions. The construct looks like this
and contains a neo cassette that interrupts gene expression followed by various reporters flanked by
FRT sites with some things inverted.
Briefly, explain how you get each color.
For all events, the neo-cassette must first be excised. For RFP you need excision of the GFP/YFP cassette,
and then no inversion. For CFP you need excision of GFP/YFP and then inversion. For YFP, you need
inversion of the first cassette. GFP requires only the excision of the neo cassette.
Given that there are variants of the loxP sites that are mutually exclusive, can you think of another way
to build a similar construct that would allow for two separate colors to be expressed stochastically?
On the left side of the construct, use both sites and then further along flank with only one or the other.