Final Exam Review Flashcards by Karah Terry

Which of the following statements is
INCORRECT?

ZPA transplanted to the posterior part of the
limb leads to duplication of digits

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The purpose of the Shh-expressing cell
transplantation experiment is to show that

Shh is expressed in the right place and at the right time for limb development

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The Shh mutant limbs contain only
one digit, which digit is it?

1 (thumb)

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A piece of foil inserted between IM and LPM
prevents limb induction because it…

Stops a secreted inducer from reaching LPM

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What do you expect in a mouse
carrying an IM-specific Fgf8 mutation?

No limb forms

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How Does Shh Induce Motor Neurons?

Model 1: Shh directly induces motor
neurons at relatively low concentration

Model 2: A secondary signal produced by
the floor plate induces motor neurons
through a relay mechanism

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Which of the following is INCORRECT?

(A) FP is closer to the source of Shh than MN

(B) It takes more Shh to induce FP than MN

(D) MN can be induced by Shh in the absence of FP

After being induced by Shh, FP produces a secondary
signal that induces MN

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What happens in Shh target cells?

Shh binds its receptor, Ptch1, on the
surface of the target cell
Smo, another cell surface protein of the
target cell, is activated
Gli transcription factor is activated and
enters the nucleus
Gene transcription is activated

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Will ectopic expression of Shh induce
an ectopic floor plate in Smo mutant
spinal cord? Why?

No, it will not!

Without Smo, the cells do not respond to
Shh, thus ectopic expression of Shh cannot
induce the formation of a floor plate.

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A chimera analysis can tell us…

(A) Shh forms a simple gradient in the spinal
cord

(B) A moderate concentration of Shh is
important for the formation of motor neuron

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Which of the following is the earliest event in the limb development?

Fgf8 expression in intermediate mesoderm

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A bead pre-soaked in saturated Shh solution
is implanted into the anterior part of a chicken
limb bud for 4 hours before it is removed.
Which extra digit can it induce?

Digit 2

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In the lineage tracing experiment I described
today, why is lacZ expression restricted to
Shh-expressing cells and their descendants?

lacZ proteins need to interact with a protein that is only present in Shh-expressing cells and their descendants.

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In the lineage tracing experiment, I described
today, why does lacZ continue to be expressed
in cells long after Shh expression stops?

Cre is very stable and continues to activate lacZ expression after Shh expression is terminated

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What regulates transcription?

Proteins:
- Transcription factors: Bind DNA
  activators and repressors
- Transcription cofactors: Do not bind DNA
  co-activators and co-repressors
DNA sequences:
- Enhancer: Bind activators
- Operator: Bind repressors

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Cis-regulatory elements:

Have to be on the same chromosome to regulate gene transcription, enhancers and operators (DNA elements)

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Trans-regulatory elements:

The genes encoding them can be on any chromosome, transcription factors and cofactors
(Proteins, RNAs)

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Which of the following is NOT a cis-
regulatory element for transcription?

(A) Promoter
(B) Enhancer
(C) Operator
(D) Transcription factor

Transcription factor

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Which of the following is NOT done to
identify a cis-regulatory element?

(A) Identify a conserved DNA sequence
(B) Drive a reporter with the conserved DNA
sequence
(C) Remove the conserved sequence to see if gene
expression is disrupted
(D) Perform a chemical mutagenesis screen to see if
the development of an animal is disrupted

Perform a chemical mutagenesis screen to see if
the development of an animal is disrupted

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Which of the following is INCORRECT about
ZRS?

A. It is a trans-regulatory element for Shh expression in ZPA.

B. Ssq insertion alters its activity

C. It is conserved between human, mouse, chicken and fish

D. It is in an intron of Lmbr1

It is a trans-regulatory element for Shh expression in ZPA.

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Which of the following is NOT
associated with RNAi?

(A) Dicer
(B) Slicer
(C) RISC
(D) DISC

DISC

What was the most important reason why
the RasGAP shRNA-expressing ES cells
was introduced into a tetraploid embryo
instead of a diploid embryo?

(A) To save time
(B) To save money
(C) Tetraploid embryos are bigger
(D) A chimera may not give birth to a mutant
pup

A chimera may not give birth to a mutant pup

Which of the following is present in the
Smo morphant, but not mutant fish?

(A) Short, curly tail
(B) Abnormally-shaped somites
(C) Increased cell death in the head
(D) Decreased p53 expression

Increased cell death in the head

Why do the Egfl7 morphants and
mutants show different phenotype?

(A) The morpholino induces non-specific cell
death
(B) The mutation in Egfl7 is not a null
(C) The upregulation of Emi proteins in the
mutant compensate for the loss of Egfl7
(D) The morpholino can target genes other than
Egfl7

The upregulation of Emi proteins in the
mutant compensate for the loss of Egfl7

Why do we clone animals?

To demonstrate that all cells have the same genetic information To study certain aspects of biology only cloned animals can provide the answer To use as a genetic tool for developmental biology study

How do we clone animals?

Somatic cell nuclear transplantation (SCNT)

An animal can be cloned through SCNT because all cells in the animal have the same...

DNA content

What is application 1 of animal cloning and how do they work?

Application 1: Genetic modification in non-Genetic Animals Mice and rats are the only species in which fully functional ES cells have been isolated Somatic cell culture, gene-targeting or other mutagenesis manipulations Once a mutant cell is identified, make a cloned animal from that cell Example: Gene-targeted Sheep - Perform gene-targeting in sheep fibroblasts - Transfer targeted fibroblast nucleus into an enucleated egg - A gene targeted lamb is born

What is application 2 of animal cloning and how do they work?

Cross-Species Cloning Nuclei from (recently) dead animals can be used for animal cloning Cross-species SCNT can be performed Fibroblasts were isolated from a recently dead mouflon. The nucleus from a fibroblast was transplanted into a sheep oocyte. A cloned mouflon was born

What is application 3 of animal cloning and how do they work?

Therapeutic Cloning

The production of a gene-targeted sheep contains all the following steps EXCEPT...

Transfer the blastocysts into a pseudopregnant sheep

Which of the following did NOT happen?

Overexpression of Pax7 leads to ectopic Dbx1 expression in the ventral spinal cord

Which of the following suggests that Shh spreads more easily through mPtc1Dloop2- expressing cells?

Pax6 expression is inhibited on the dorsal side of patches of mPtc1Dloop2-expressing cells Nkx2.2 expression is dorsally expanded on the dorsal side of patches of mPtc1Dloop2-expressing cells

Where is AER?

at the distal edge

Which of the following is NOT true about Shh? (A) Shh-expressing cells induce digit duplication when they are grafted to the anterior edge of the limb bud (B) Shh is expressed in the floor plate and notochord of a mouse embryo (C) Shh is required for the formation of all digits except for the thumb/big toe (D) Shh is expressed in the AER and promotes the outgrowth of the limb buds

When an FGF4 bead is implanted to the mesoderm in the interlimb region, an extra limb develops. What does this tell you?

One of the FGF family members must be expressed there

Which of the following results in Shh expression in the anterior part of the limb bud? (A) RA beads, no AER (B) FGF8 beads, no AER (C) RA beads, with AER (D) FGF8 beads, with AER

Which of the following is NOT true? (A) A moderate level of Shh can induce motor neurons in a spinal cord explant (B)The spinal cord explant experiment addresses whether motor neuron is induced by Shh in vivo (C) Smo mutant cells fail to become motor neurons in a chimera (D) The chimera analysis does NOT address whether motor neurons form at a specific Shh concentration

Which of the following was observed when Shh beads were implanted into the anterior part of the chicken limb buds? (A) Beads containing higher concentration of Shh induce more posterior digits (B) Beads have to be in the limb buds for at least 16 hours to induce extra digits (C) It takes longer time to induce the posterior digits than the anterior ones (D) All of the above

Which of the following is INCORRECT about cis-regulatory elements for gene expression? (A) They can only regulate the expression of genes on the same chromosome (B) They have to be right next to the promoters of the genes they regulate (C) They usually associate with proteins that regulate transcription of the gene (D) Their sequences are usually conserved during evolution

B is false because A is true because it is cis meaning same in this case -if it was trans it could regulate other chromosomes C is true because D is true because

Which of the following is NOT true about miRNAs? (A) They are short double-stranded RNAs (B) They mainly interfere with protein translation (C) DICER is important in the processing of miRNAs (D) miRNAs mainly target virus RNAs for degradation

Which of the following is NOT part of a transgene expressing shRNA? (A)A RNA polymerase II-based promoter (B)A 7-base spacer between the sense and antisense sequence (C) Multiple Ts at the end of the transcript (D) A palindromic sequence matching the target gene

The Morpholino knockdown of Egfl7, but not a mutation in Egfl7 gene in the genome, disrupts blood vessel network because... (A)The morpholino knockdown causes non-specific effect (B) The mutation in Egfl7 is a hypomorph (C) The increase in emi proteins compensate for the loss of Egfl7 in mutants, but not in morphants (D) The increase in emi proteins compensate for the loss of Egfl7 in morphants, but not in mutants

An siRNA against lacZ results in which of the following in zebrafish (A) The reduction of the expression of a lacZ transgene (B) The reduction of the expression of many developmentally important genes (C) Defects in the heads and tails (D) All of the above

Which of the following about animal cloning is INCORRECT? (A)A cloned animal is exactly the same as the nucleus donor (B)Animal cloning works because the same genetic information exists in the chromosomes of all cells in an animal (C) During cloning, proteins in the cytoplasm of the oocyte help to erase the chromosomal modifications on the donor nucleus (D) The cloning efficiency correlates inversely with the age of the nucleus donor

Which of the following is INCORRECT? (A) The purpose of therapeutic cloning is to derive ES cells from cloned embryos (B) After ES cells are derived from cloned embryos, they can be transplanted back into the patient directly (C) Therapeutic cloning is expected to minimize immunorejection of transplanted cells (D) Caffeine is critical for the success of therapeutic cloning

Which of the following about Yamanaka Factors is NOT true? (A)They can convert skin cells into stem cells (B)They can cause tumor (C)They are hydrophobic proteins that can easily cross the cell membrane (D) Their stem cell inducing effect can be mimicked by small chemicals

Inducing pluripotent stem cells with chemical compounds... (A) Is less expensive than the original Yamanaka method (B) Is faster than the protein-based induction method (C) May disrupt endogenous genes (D) May turn the cells into tumor cells

Essay 1: Based on the temporal gradient theory, what kind of phenotype do you expect to see in mouse limb development if Shh is mutated at E11.5?

According to this theory, the posterior digits require longer exposure to Shh than anterior digits. Therefore, if Shh is mutated at E11.5, the fifth digit may not form. Digits 3 and 4 should be normal as they no longer express Shh at this stage.

Essay 2: Name two methods to ensure the phenotype you observed in siRNA or morpholino knockdown animals are NOT due to off-target effects.

method 1: If you can rescue the phenotype by re-introducing the target gene, the phenotype is likely specific. method 2: If you do not see the same phenotype when you introduce a scrambled siRNA or morpholino, the phenotype in the knockdown animal is likely specific. Additional methods you can mention: perform the knockdown in p53 mutants, perform the knockdown in a KO animal, etc

Essay 3: Describe how you can generate a mutant farm animal for which ES cells do not exist and it is still too expensive to take the CRISPR/Cas9 approach and screen hundreds of animals.

You can take the approach described in the lecture. First, you culture skin fibroblasts from an adult animal, and generate mutant cells through gene-targeting or CRISPR/Cas9. Then you produce a cloned animal by transplanting the mutant nucleus into an enucleated oocyte. A pseudo-pregnant female will be needed for the oocyte to develop to term.