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Biology 3338A Lecture 2: Developmental Biology Section 2

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School
Western University
Department
Biology
Course
Biology 3338A
Professor
Sashko Damjanovski

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Document Summary

Chimeric mouse: an animal made up of different things. A mouse that"s made up of different cells; mixture of different cells; not its own cells. Transgenic mouse: carrying genes/mutations that it doesn"t have before. Mouse inner cell mass (icm) cells are toti; not specified; can be cultured, amplified and returned into a trophoblast whichcan then be reared in a host mother. If you moved the icm cells around, it doesn"t matter because they are not specified and they can be cultured. You can take the cells out and grow millions of them in vitro in the same stage (they will just divide but stay at the same developmental stage) You can even put them back into the icm of another embryo and back into the uterus of a mouse and it will continue to grow into a normal mouse. You can use transgenic mice as tools to understand medicine; you can make mice that lack genes or have extra genes.

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Related Questions

Summary

please!

Multipotent versus restricted

Morphologically detectable neural crest cells usually are first observed as the cells individualize and delaminate upon emigration from the neural tube, following their epithelial to mesenchymal transition (EMT), in which they convert from a tightly adherent sheet of cells to a dispersed and more individual mesenchymal population. Prior to EMT, however, it is difficult or impossible at most axial levels and in most species to distinguish presumptive neural crest cells from cells that will form dorsal neural tube derivatives. However, there are exceptions, such as a subpopulation of cranial neural crest cells in chick and mouse that appears to be set aside and morphologically distinct at midbrain levels. In addition, some species such as axolotl have a clearly segregated population of neural crest cells that exists as a ridge on the dorsal neural tube.

A long-standing debate in the neural crest literature has been whether neural crest cells are multipotent and/or restricted in their developmental potential. In other words, can a single neural crest precursor form only one type of derivative or are the cells multipotent and able to produce multiple derivatives. Single cell lineage experiments (Bronner-Fraser and Fraser, 1988), in which individual cells within the chick dorsal neural tube are labeled with vital dye, show that some of the labeled clones contribute to multiple differentiated cell types in the periphery, including melanocytes, sympathetic and sensory ganglion cells. Thus, the original precursor was “multipotent” in its developmental potential to form neural crest derivatives. In addition to neural crest derivatives, single dorsal neuroepithelial cell also give rise both to migrating neural crest cells and cells that remain in the dorsal neural tube, such as roof plate cells and dorsal sensory neurons and/or interneurons. This suggests a shared lineage between the neural tube and neural crest, at least at this stage. However, labeling migrating neural crest cells in vivo also produced clones that could contribute to more than one neural crest lineage (Fraser and Bronner-Fraser, 1991), again supporting the idea that some of the migrating population retained multipotency.

Clonogenic culture of neural crest cells cultured shortly after their emigration from the neural tube definitely shows that many early migrating neural crest cells are multipotent in vitro as well (Baroffio et al., 1988, Calloni et al., 2007, Calloni et al., 2009, LeDouarin et al., 2008a, LeDouarin et al., 2008b, Sieber-Blum and Cohen, 1980 and Stemple and Anderson, 1993). Exposure to different growth factors can profoundly influence their lineage decisions (e.g. Lahav et al., 1998). Furthermore, they have a capacity for self-renewal, at least for a few cell divisions (Stemple and Anderson, 1993 and Trentin et al., 2004).

The fact that individual neural crest cells can form multiple derivatives has led to the idea that they have stem cell properties. Stem cells are defined as individual progenitor cells that can generate one or more specialized cell types. A cardinal feature of stem cells is their ability to self-renew, that is, to divide so as to give rise to at least one daughter cell that maintains the multipotent character of its parent. The fact that cloned neural crest cells have a limited ability to self-renew has led to the idea that they are stem-like (progenitors) cells rather than true stem cells. Interestingly, however, neural crest stem cells can be derived from adult tissues (Fernandes et al., 2008 and Shakhova and Sommer, 2010; see Chapter Dupin and Sommer), suggesting that they can remain quiescent for long periods of time or maintain long term self-renewal ability when left in situ.

The presence of some multipotent neural crest precursors cannot, however, rule out the possibility that other precursors may be more restricted in their developmental potential. In fact, experiments in zebrafish suggested that neural crest cells contribute to different sets of derivatives accordingly to their migration order (Raible and Eisen, 1994). However, if the leader cell was ablated, the next cell in line took up the fate that would have been filled by the ablated cell (Raible and Eisen, 1996). Similarly, early migrating neural crest cells normally exhibit a broader range of derivatives than later migrating cells; however, when the early population is ablated and replaced by late migrating cells, the late migrating cells assume a broader developmental potential than that prescribed by their normal fate (Baker et al., 1997). This raises a very important issue: that developmental potential is greater than or equal to a cell's normal fate. Only by challenging the cell by putting it into a new environment can one test for restriction of cell fate. This is best exemplified by experiments in which the potential of neural crest populations was challenged by performing heterotopic transplants between different axial levels (LeDouarin and Teillet, 1974), such as exchanging cranial and trunk, or vagal and trunk populations. The results demonstrate a combination of flexibility in cell fate and some axial level-autonomous characteristics. For example, cranial neural crest cells normally make cartilage and bone of the face whereas trunk neural crest cells do not. Transplantation of cranial neural folds to the trunk results in production of many normal trunk derivatives, as well as the formation of ectopic cartilage nodules (Le Lievre et al., 1980 and LeDouarin and Teillet, 1974). Conversely, transplantation of trunk neural folds to the head results in contributions to cranial neurons and glia of cranial ganglia, but not to cartilage, although some connective tissues and pericytes derive from this graft (Nakamura and Ayer-le Lievre, 1982). This reveals some flexibility in fate, but a more limited ability to form skeletal derivatives (LeDouarin et al., 2004). However, trunk neural crest cells can form cartilage in vitro under appropriate culture conditions (Calloni et al., 2007 and McGonnell and Graham, 2002). Because challenging prospective neural crest fate is a difficult experiment, it is much easier to prove multipotency than restricted cell fate, leaving the question of whether or not there are lineage-restricted neural crest precursors still open to debate (see Krispin et al., 2010).

HELP ASAP; Please help me understand my lab results, so be detailed to clearly understand

Then , Please include answers to the question below to include in your response

1. what role does ampicilin (amp) and arabinose (ara) play in the experiment.

2) Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes). In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes. The pGLO plasmid contains the gene for GFP, and also contains the gene for the enzyme β-lactamase (bla), which provides resistance to the antibiotic ampicillin. The β-lactamase protein is produced and secreted by bacteria that contain the plasmid. β-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express β-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. 1) Cells are first treated with a solution of CaCl2, which is thought to neutralize the repulsive negative charges of the phosphate backbone of DNA and the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to making the cells ‘competent’ (i.e. the cells are capable, or competent, to take-in exogenous DNA). 2) Cells are then subjected to a heat shock, which is thought to increase the permeability of the cell membrane, allowing the cells to take-in the plasmid DNA. 3) Cells are allowed to grow in a ‘recovery’ phase in the absence of ampicillin. During this time the cells begin to produce the β-lactamase protein, which will allow them to survive when they are subsequently placed on agar plates containing ampicillin.

For the first week section of lab

To eac

h of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate. Transfer this colony to one of the microfuge tubes.

Gently vortex the tube until the colony is dispersed. Repeat for the second tube. Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes. Incubate both tubes at 42°C for 50 seconds.

Incubate both tubes on ice for 2 minutes. Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes. Flick each tube gently. Add 100ul of t

his transformation mix to the appropriate plates.

Results For plates:

+DNA: Has the E. Coli pGlo plasmid and - DNA plates do no have EColi pGLo plasmid .

Amp means ampiclin and Ara means arabinose on agar plates

+ DNA Plate LB/amp/ara--> white 9 colonies, Glow: yes

+DNA LB/amp--> 7 colonies, GLow: no

--DNA LB/AMP- 1 colony so no colony growth and no glovw

-DNA: LB--> many colonies, Glow: no

Please explain significance of result or thand the reason for the results in scientific terms based on the background information provided and your own knowledge to be detailed. Would these results be expected as wll.

Expression vectors inprokaryotes do not make functional eukaryotic gene products inbacteria very well because

Answer Not sure which one ?!

· the codon sequence for prokaryotes is differentthan the codon sequence in eukaryotes

· there are no disulfide bridges formed in proteinsnormally made in prokaryotes

· prokaryotic expression vectors cannot translateeukarytic sequences

· eukaryotic genes have introns, and prokaryotesdon't

· eukaryotic genes have exons and prokaryotesdon't


I create a knockout mouseusing the agouti/black fur system \. When I cross the agoutioffspring of the originally engineered mouse, I find a ratio of 2agouti mice to 1 black furred mouse. What is the bestexplanation?

Answer not sure which one?!

· The gene knocked out was recessive.

· The gene knocked out was dominant.

· The gene knocked out was a lethalgene.

· The knockput was integrated into a random spot,and did not knockput the original gene.

A restriction enzyme cuts DNAand leaves the following end

xxCTGCA
xxG

Which of the following could be the sequence of the correspondingend of the other end of the cut DNA?

Answer

· xxG
xxCTGCA

· xxC
xxGTGCA

· xxCCGAT
xxG

· xxGGCTA
xxC


An SNP always occurs dueto

Answer

· a one base-pair change.

· a three base-pair change.

· a single restriction site change.

· a single amino acid change.

· None of these.


I PCR out a mutated gene in apatient with Lisenbee chorea (the inability to dance in acoordinated fashion) and I compare it to another patient with thatsame disease phenotype. One subject had a mutation on chromosome 4,and the other subject couldn't rock it because of a mutation onchromosome 5. This is an exampe of

Answer

· pleitropy

· locus heterogeneity

· allelic heterogeneity

· dominant negative mutation

· penetrance

In his experiments, Mendelnoted that when two traits are involved in a genetic cross, theyare inherited independently of each other. Though Mendel didn'tknow about chromosomes, this still holds true (mostly)because

Answer

· genes on the same chromosome are often mixedthrough meiotic crossover during the formation ofgametes.

· variant alleles of the same gene do not interferewith the phenotypic expression of the gentoype in different geneticloci.

· most higher organisms are diploid, making only twoalleles available for every genetic locus, and there is a 50/50chance of geting either allele from a single parent.

· expression of phenotypes can vary from thegenotypes because of encironmantal influences.

· chromosomes often recombine

Anticipation is caused by amutation that increases in expressivity over subsequentgenerations.

Answer

· True

· False

Question 44

I have a genotype that shouldproduce a specific phenotype, but some of the individuals with thegenotype do not demonstrate any evidence of the phenotype. I wouldconsider this an example of

Answer

· allelic heterogeneity.

· locus heterogeneity.

· penetrance.

· expressitivity.

· sheer luck.

Question

Which of the following doesnot occur during the PCR reactions?

Answer

· Changes in the reaction temperature by athermocycler

· Denaturation at high temperatures

· Synthesis of oligonucleotide primers

· Extension of primers by DNA polymerase

· Annealing of oligonucleotide primers

Question

DNA markers, or variantnon-coding regions of DNA, are DNA polymorphisms that are usefulfor genetic mapping.

Answer

· True

· False

A mosaic is an organismwith

Answer

· multiple genotypes within one organism

· multiple alleles within one genotype

· more than one color of fur

· transgenes added to the zygote beforedevelopement

· a wt phenotype but a mutated genotype

Question

Genotype causesphenotype.

Answer

· No, gentoype just influences phenotype.

· Yes, genotype is the DNA sequence that createsphenotype.

Question

A true genetic chimera can becreated by

Answer

· mutating a gene early on in the development of anorganism resulting in different alleles being present in theadult

· multiple fertilized eggs or zygotes fusing to formone embryo

· adding a transgene to the genome of an organismduring fetal development only

· adding cells of a different species to an adultorganism

· adding a transgene to the genome of an animal atany stage in development

Question

The ABO blood group can bestbe explained by the concept of _______.

Answer

· dominant traits

· recessive traits

· allelic heterogeneity

· locus heterogeneity

· vampirism

If a loss of functionmutation creates a dominant phenotype, it may be becauseof

Answer

· haploinsufficiency

· penetrance

· expressivity

· allelic heterogeneity

· locus heterogeneity

Please select the best matchfor each.

Answer

· PCR: Technique used to generatecopies of a DNA fragment

· Southern blot : Screeningprocedure used to assay DNA fragments

· RFLPs : DNA fragments generatedby restriction enzymes that vary in length betweenindividuals

· Northern blot : Screeningprocedure used to assay mRNA fragments

· Electrophoresis Movement ofmacromolecules in an electrical field

Answer

A.

Screening procedure used toassay DNA fragments

B.

Movement ofmacromolecules in an electrical field

C.

Screening procedure used toassay mRNA fragments

D.

DNA fragments generated byrestriction enzymes that vary in length betweenindividuals

E.

Technique used to generatecopies of a DNA fragment

A gene mutates, and theprotein produced has a novel way of interacting with the cell, andcreates a new phenotype because of this new functionality. This iscalled

Answer

· pleitropy

· locus heterogeneity

· allelic heterogeneity

· dominant negative mutation

· gain of function dominant mutation

Question 62

Mutations in the somaticchromosomes may be inherited by the next generation.

Answer

· True

· False


A degenerate PCR primer withmany variant sequences must be used to make multiple copies of agene

Answer

· if only the protein sequence of the gene productis known to construct the primers

· if the DNA you are using is cDNA to constructthe primers

· if the DNA you are using is genomic DNA to becopied

· if the DNA you are trying to copy iscDNA

· if the vector is prokaryotic and the transformedcell is eukarytotic

Question

Please select the best matchfor each term.

Answer

· Restriction enzyme


· PCR C

· Plasmid

· YAC A

· Dideoxynucleotide

Answer

A.

Endonuclease isolated frombacteria

B.

Eukaryotic cloningvector

C.

Method for amplification ofDNA sequences

D.

Small circular fragment ofDNA

E.

Molecule used in DNAsequencing

Question

A couple goes on MauryPovitch, and the results are in: you are not the father. But noother man impregnated the female (granted, unlikely for a MauryPovitch guest, but work with me here) and he must be the father.DNA analysis claims otherwise, though the child definitely wasmom's (poor thing). What may be going on here?

Answer

· the child is parthenogenic because the motheractually impregnated herself like a shark, and the child's DNA isall mom's

· the child had a mutation that changed the genethat is used to trace paternity

· the child is a mosaic because he is actually a setof twins fused early during fetal development, and therefore camefrom 2 eggs and 2 sperm

· the dad may have germ-line mosaicism, meaning thatthe genotype of his sperm is different from his somaticgenotype

· mitochondrial DNA only comes from mom, so there isno way to tell whobthe baby's father is