Developmental Exam I

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How can you birthdate a neuron?

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How can you birthdate a neuron?

add a molecule that gets added during replication or use antibodies to tag PCNA

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tagging PCNA vs molecule replication

PCNA is only good short term and antibodies are species specific and molecule replication used to be radioactive

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Lineage Analysis vs Birthdating

birthdating tags new neurons while lineage analysis provides a family tree

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How does lineage analysis with a retrovirus work?

a cell is infected with a modified retrovirus which replenishes with division tagging new cells

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Pros and Cons of retrovirus

a pro is that it contains a reporter gene but it cannot replicate

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Retrovirus Examples

Horseradish peroxidase and B-Galactosidase

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How does lineage analysis with Cre-LoxP work?

cre is an enzyme that causes recombination at a floxed DNA site

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what are major challenges to studying gene expression?

  • multiple time points to be studied

  • Neuroplasticity

  • diverse cell populations intermingle

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what methods can find DNA anatomically?

Immunohistochemistry

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What can you learn using immunohistochemistry?

where protein is expressed using primary and secondary antibodies

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Pros and cons of immunohistochemistry

it provides spatial information but antibodies have to be made and are species specific

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what methods can find DNA molecularly?

Western blot and Elisa

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what can you learn using western blot?

the quantity of protein expressed

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Pros and Cons of western blot

It comes in a large quantity but gives no spatial info and needs antibodies

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How is Elisa used?

there is a 96 well plate lined with antibodies where proteins are read

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What method can find mRNA anatomically?

In Situ Hybridization

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what does ISH tell you?

where mRNA is expressed

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Pros and Cons of In Situ Hybridization

It provides spatial info and has a wide range of probes but the probes are specific

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What methods can find mRNA molecularly?

Northern Blot and qRT-PCR

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What is northern blot used for?

to figure out if mRNA is present or not

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Pros and Cons of Northern Blot

the cost is low and its fast, but there is no spatial info

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what does qRT-PCR tell you

the specific quantity of mRNA

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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

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what is a primary antibody?

It finds a proteins

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what is a secondary antibody?

it finds the primary and reveals it

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Carnegie Stage 1

fertilization of the egg

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Carnegie Stage 2

first cleavage through morula

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Carnegie Stage 3

unimplanted blastocyte

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what does the inner cell mass become?

the embryo

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what does the outer cell mass become?

the placenta

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when do the inner and outer cell mass form?

Carnegie Stage 3

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Carnegie Stage 4

shedding of zona pellucida

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Carnegie Stage 5

implantation is complete

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Carnegie Stage 6

Gastrulation begins and the primitive steak appears

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When do the 3 germ layers form?

Carnegie Stage 6

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What are the 3 germ layers?

endoderm, mesoderm, ectoderm

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Carnegie Stage 7

cell migration defining gastrulation

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what does the endoderm turn into?

insides and organs

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what does the mesoderm turn into?

muscles

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what does the ectoderm turn into?

skin and nervous system

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Carnegie Stage 8

appearance of neural plate and neural groove

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Carnegie Stage 9

somites appear on either side of the neural groove

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what are somites?

precursors of dermal tissue, muscles, vertebrae

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Carnegie Stage 10

formation of neural tube

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Carnegie Stage 11

more somites grow

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Carnegie Stage 12

neural tube closes

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Carnegie Stage 13

30 pair of somites

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Carnegie Stage 14

rapid brain growth with definition of forebrain, hindbrain, and midbrain

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Carnegie Stage 15

cerebral hemispheres can be discerned

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what are the three primary brain vesicles?

prosencephalon, mesencephalon, and rhombencephalon

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which of the primary brain vesicles subdivide further?

prosencephalon and rhombencephalon

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what does the prosencephalon divide into?

the telencephalon and diencephalon

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what does the rhombencephalon divide into?

metencephalon and myelencephalon

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What does the telencephalon form?

cerebrum

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what does the diencephalon form?

eye cups and thalamus

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what does the metencephalon form?

pons and cerebrum

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what does the mesencephalon form?

midbrain

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what does the myelencephalon form?

medulla oblongata

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What defects are caused by the failure to close the anterior/cranial neuropore?

iniencephaly, encephalocele, and anencephaly

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characteristics of iiencephaly?

“stargazing” posture with a stillbirth or premature death

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Characteristics of encephalocele?

herniated meninges and brain tissue

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Characteristics of anencephaly

total or partial absence of brain

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What failed to close in craniorachischisis?

middle portion

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Characteristics of craniorachischisis?

anencephaly along with lesion on spinal cord with “stargazing” look

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what failed to close in spina bifida?

posterior/caudal neuropore

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Characteristics of spina bifida

herniation of meninges and spinal cord

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mutation

change in DNA sequence

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null mutation

complete failure to express protein coded by mutated gene

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hypomorph

wild type gene expressed at a low level

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wild type

standard, unmutated gene

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strain

a reproductively isolated group of animals

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inbred strain

over 20 consecutive generations of sibling mating to produce homozygosity

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isogenic

strain where every individual is genetically identical

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What is forward genetics?

identifying a gene then finding the phenotype

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What is reverse genetics?

selecting a gene to then find a phenotype

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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

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What committee oversees institutional use of animals

Institutional Animal Care and Use Committees (IACUC

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Theiler Stage 1

fertilization

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Theiler Stage 7

blastocyst sheds zona pellucida and implants

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Theiler Stage 11

appearance of neural plate and neural groove

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Theiler Stage 12

portion of neural tube closes

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Theiler Stage 14

anterior portion of tube closes

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Theiler Stage 15

posterior neuropore forms

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Theiler Stage 16

posterior neuropore closes

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