CMMB 403 final exam
✅✅
what does CRISPR use in order to generate changes in genome? is CRISPR
forward or reverse genetics? - -- nuclease
- 20 nucleotide RNA sequence to target specific sites in the genome
- reverse genetics
what is the role of Cas9 in CRISPR - ✅✅-endonuclease that catalyzes
double stranded breaks
✅✅
what are the two repair paths cells can use to repair double stranded breaks
from CRISPR-Cas9 - -1. non-homologous end joining (NHEJ)
- naturally try to repair
- imperfect, creates indels causing frameshift mutation
2. directed homology repair
- sequence homology on 5' and 3' ends to encourage homologous
recombination flanking the double stranded breaks
- insert known sequences to try and treat genetic diseases
explain the method of conditional gene inactivation - ✅✅ -- make targetting
construct in lab
- electroporate construct into embryonic stem cells to target endogenous
Shox2 locus
- mice generated by blastocyst injection into ESCs carry loxP sites therefore
can delete Shox2
2 components for a conditional knockout mouse - ✅✅ -1. transgenic mice
express Cre recombinase in the tissue of interest
2. mice with loxP sites on each side of the gene or region to be deleted
✅✅
How is CRISPR-Cas9 being used in a patient in the USA to treat sickle cell
anemia? - -by inactivating an enhancer of the BCL11A gene
(BCL11A gene represses fetal hemoglobin)
how does the BCL11A protein affect global gene expression - ✅✅-it
represses fetal hemoglobin gene expression
,In what kind of animals is CRISPR good? - ✅✅-any animals for which one
cell embryos are accessible
- good for mammals, not chickens
what is the result of CRISPR inactivating Rspo2 in half a frog? - ✅✅-Rspo
knockout= limbless
side inactivated by CRISPR had no limb
what are transgenic mice good for?
- how are they made?
✅✅
- is this targeted?
- what are they not useful for? - -good for mis-expressing genes,
enhancer reporters, etc.
- generated by pronuclear injection; DNA construct is incorporated randomly
into the genome
- not useful for LOF because not targeted
how are knockout mice made?
✅✅
- is it targeted?
- what kind of species is this successful in? - -created by genomic
targeting in embryonic stem cells
- DNA construct is targeted to specific sites in the genome by homologous
recbomination
- mice then made from pluripotent ES cells
- only successful in mice
✅✅
what is CRISPR/Cas9 good for?
- what kind of species is it successful in? - -efficient RNA-directed
targeting in single-cell embryos or cell lines from a broad variety of species
- useful for knockout, introducing changes in genome, corrections, etc.
- many species
when does organogenesis occur in human development? - ✅✅-4th-8th
week
what day is the neural plate formed? - ✅✅-day 18
what day does the primitive streak disappear? - ✅✅-day 25
, ✅✅
what is responsible for neural induction? where are these signals from? -
-by BMP inhibitors from the pharyngeal endoderm, prechordal plate and
notochord mesoderm
✅✅
on what day does the neural plate consist of columnar cells that are taller than
surrounding cells? - -day 20-21
✅✅
during what week does the embryo fold into a 3D structure, most organs start
to form, heart starts beating and neural tube forms? - -4th week
- germ disc starts to look more like a body
on what day does the neural tube close? - ✅✅-day 28
anencephaly - ✅✅-caused by failure of the anterior neuropore to close
- fatal
craniorachischisis - ✅✅-failure of the entire neural tube to close
- fatal
✅✅
spina bifida - -caused by failure of the posterior neuropore to close
- can be surgically fixed
what are the 3 major divisions of the ectoderm germ layer? - ✅✅-1. surface
ectoderm (epidermis)
2. neural crest
3. neural plate/neural tube
T or F: all 3 major subdivisions of the ectoderm can form neurons - ✅✅-true
what structures does the surface ectoderm form? - ✅✅-- epidermis
- sensory epithelium
- lens/cornea
- etc
what structures does the neural crest form? - ✅✅-- peripheral nervous
system (PNS)
- melanocytes
- facial cartilage
- adrenal medulla
- Schwann cells
✅✅
what does CRISPR use in order to generate changes in genome? is CRISPR
forward or reverse genetics? - -- nuclease
- 20 nucleotide RNA sequence to target specific sites in the genome
- reverse genetics
what is the role of Cas9 in CRISPR - ✅✅-endonuclease that catalyzes
double stranded breaks
✅✅
what are the two repair paths cells can use to repair double stranded breaks
from CRISPR-Cas9 - -1. non-homologous end joining (NHEJ)
- naturally try to repair
- imperfect, creates indels causing frameshift mutation
2. directed homology repair
- sequence homology on 5' and 3' ends to encourage homologous
recombination flanking the double stranded breaks
- insert known sequences to try and treat genetic diseases
explain the method of conditional gene inactivation - ✅✅ -- make targetting
construct in lab
- electroporate construct into embryonic stem cells to target endogenous
Shox2 locus
- mice generated by blastocyst injection into ESCs carry loxP sites therefore
can delete Shox2
2 components for a conditional knockout mouse - ✅✅ -1. transgenic mice
express Cre recombinase in the tissue of interest
2. mice with loxP sites on each side of the gene or region to be deleted
✅✅
How is CRISPR-Cas9 being used in a patient in the USA to treat sickle cell
anemia? - -by inactivating an enhancer of the BCL11A gene
(BCL11A gene represses fetal hemoglobin)
how does the BCL11A protein affect global gene expression - ✅✅-it
represses fetal hemoglobin gene expression
,In what kind of animals is CRISPR good? - ✅✅-any animals for which one
cell embryos are accessible
- good for mammals, not chickens
what is the result of CRISPR inactivating Rspo2 in half a frog? - ✅✅-Rspo
knockout= limbless
side inactivated by CRISPR had no limb
what are transgenic mice good for?
- how are they made?
✅✅
- is this targeted?
- what are they not useful for? - -good for mis-expressing genes,
enhancer reporters, etc.
- generated by pronuclear injection; DNA construct is incorporated randomly
into the genome
- not useful for LOF because not targeted
how are knockout mice made?
✅✅
- is it targeted?
- what kind of species is this successful in? - -created by genomic
targeting in embryonic stem cells
- DNA construct is targeted to specific sites in the genome by homologous
recbomination
- mice then made from pluripotent ES cells
- only successful in mice
✅✅
what is CRISPR/Cas9 good for?
- what kind of species is it successful in? - -efficient RNA-directed
targeting in single-cell embryos or cell lines from a broad variety of species
- useful for knockout, introducing changes in genome, corrections, etc.
- many species
when does organogenesis occur in human development? - ✅✅-4th-8th
week
what day is the neural plate formed? - ✅✅-day 18
what day does the primitive streak disappear? - ✅✅-day 25
, ✅✅
what is responsible for neural induction? where are these signals from? -
-by BMP inhibitors from the pharyngeal endoderm, prechordal plate and
notochord mesoderm
✅✅
on what day does the neural plate consist of columnar cells that are taller than
surrounding cells? - -day 20-21
✅✅
during what week does the embryo fold into a 3D structure, most organs start
to form, heart starts beating and neural tube forms? - -4th week
- germ disc starts to look more like a body
on what day does the neural tube close? - ✅✅-day 28
anencephaly - ✅✅-caused by failure of the anterior neuropore to close
- fatal
craniorachischisis - ✅✅-failure of the entire neural tube to close
- fatal
✅✅
spina bifida - -caused by failure of the posterior neuropore to close
- can be surgically fixed
what are the 3 major divisions of the ectoderm germ layer? - ✅✅-1. surface
ectoderm (epidermis)
2. neural crest
3. neural plate/neural tube
T or F: all 3 major subdivisions of the ectoderm can form neurons - ✅✅-true
what structures does the surface ectoderm form? - ✅✅-- epidermis
- sensory epithelium
- lens/cornea
- etc
what structures does the neural crest form? - ✅✅-- peripheral nervous
system (PNS)
- melanocytes
- facial cartilage
- adrenal medulla
- Schwann cells
