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How can you birthdate a neuron?
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add a molecule that gets added during replication or use antibodies to tag PCNA
tagging PCNA vs molecule replication
PCNA is only good short term and antibodies are species specific and molecule replication used to be radioactive
Lineage Analysis vs Birthdating
birthdating tags new neurons while lineage analysis provides a family tree
How does lineage analysis with a retrovirus work?
a cell is infected with a modified retrovirus which replenishes with division tagging new cells
Pros and Cons of retrovirus
a pro is that it contains a reporter gene but it cannot replicate
Horseradish peroxidase and B-Galactosidase
How does lineage analysis with Cre-LoxP work?
cre is an enzyme that causes recombination at a floxed DNA site
what are major challenges to studying gene expression?
multiple time points to be studied
diverse cell populations intermingle
what methods can find DNA anatomically?
What can you learn using immunohistochemistry?
where protein is expressed using primary and secondary antibodies
Pros and cons of immunohistochemistry
it provides spatial information but antibodies have to be made and are species specific
what methods can find DNA molecularly?
Western blot and Elisa
what can you learn using western blot?
the quantity of protein expressed
Pros and Cons of western blot
It comes in a large quantity but gives no spatial info and needs antibodies
How is Elisa used?
there is a 96 well plate lined with antibodies where proteins are read
What method can find mRNA anatomically?
In Situ Hybridization
what does ISH tell you?
where mRNA is expressed
Pros and Cons of In Situ Hybridization
It provides spatial info and has a wide range of probes but the probes are specific
What methods can find mRNA molecularly?
Northern Blot and qRT-PCR
What is northern blot used for?
to figure out if mRNA is present or not
Pros and Cons of Northern Blot
the cost is low and its fast, but there is no spatial info
what does qRT-PCR tell you
the specific quantity of mRNA
Pros and cons of qRT-PCR
it is fast and can measure a lot but there is no spatial info and it is hard to be specific
what is a primary antibody?
It finds a proteins
what is a secondary antibody?
it finds the primary and reveals it
Carnegie Stage 1
fertilization of the egg
Carnegie Stage 2
first cleavage through morula
Carnegie Stage 3
what does the inner cell mass become?
what does the outer cell mass become?
when do the inner and outer cell mass form?
Carnegie Stage 4
shedding of zona pellucida
Carnegie Stage 5
implantation is complete
Carnegie Stage 6
Gastrulation begins and the primitive steak appears
When do the 3 germ layers form?
What are the 3 germ layers?
endoderm, mesoderm, ectoderm
Carnegie Stage 7
cell migration defining gastrulation
what does the endoderm turn into?
insides and organs
what does the mesoderm turn into?
what does the ectoderm turn into?
skin and nervous system
Carnegie Stage 8
appearance of neural plate and neural groove
Carnegie Stage 9
somites appear on either side of the neural groove
what are somites?
precursors of dermal tissue, muscles, vertebrae
Carnegie Stage 10
formation of neural tube
Carnegie Stage 11
more somites grow
Carnegie Stage 12
neural tube closes
Carnegie Stage 13
30 pair of somites
Carnegie Stage 14
rapid brain growth with definition of forebrain, hindbrain, and midbrain
Carnegie Stage 15
cerebral hemispheres can be discerned
what are the three primary brain vesicles?
prosencephalon, mesencephalon, and rhombencephalon
which of the primary brain vesicles subdivide further?
prosencephalon and rhombencephalon
what does the prosencephalon divide into?
the telencephalon and diencephalon
what does the rhombencephalon divide into?
metencephalon and myelencephalon
What does the telencephalon form?
what does the diencephalon form?
eye cups and thalamus
what does the metencephalon form?
pons and cerebrum
what does the mesencephalon form?
what does the myelencephalon form?
What defects are caused by the failure to close the anterior/cranial neuropore?
iniencephaly, encephalocele, and anencephaly
characteristics of iiencephaly?
“stargazing” posture with a stillbirth or premature death
Characteristics of encephalocele?
herniated meninges and brain tissue
Characteristics of anencephaly
total or partial absence of brain
What failed to close in craniorachischisis?
Characteristics of craniorachischisis?
anencephaly along with lesion on spinal cord with “stargazing” look
what failed to close in spina bifida?
Characteristics of spina bifida
herniation of meninges and spinal cord
change in DNA sequence
complete failure to express protein coded by mutated gene
wild type gene expressed at a low level
standard, unmutated gene
a reproductively isolated group of animals
over 20 consecutive generations of sibling mating to produce homozygosity
strain where every individual is genetically identical
What is forward genetics?
identifying a gene then finding the phenotype
What is reverse genetics?
selecting a gene to then find a phenotype
Forward vs Reverse genetics
forward genetics has no bias of previous knowledge and can exploit spontaneous mutations while reverse genetics must inactivate a known gene and the mutation may not be visible
What committee oversees institutional use of animals
Institutional Animal Care and Use Committees (IACUC
Theiler Stage 1
Theiler Stage 7
blastocyst sheds zona pellucida and implants
Theiler Stage 11
Theiler Stage 12
portion of neural tube closes
Theiler Stage 14
anterior portion of tube closes
Theiler Stage 15
posterior neuropore forms
Theiler Stage 16
posterior neuropore closes