Week 11 Flashcards
(145 cards)
1
Q
How many conceptions are lost in the first 5 months?
A
23%
2
Q
How many children are born with malformations?
A
Over 5%
3
Q
Out of 20 eggs in US and Western countries how many come to term?
A
Only 6.2 are expected to come to full term
4
Q
What is the name of exgenous agents responsible for birth defects?
A
Teratogens
5
Q
What are examples of teratogens?
A
Drugs, chemicals, ionising radiation, high temperature, infectious micro-organsisms and metabolic conditions in the mother
6
Q
When are embryos most susceptible to teratogens?
A
During the embryonic period between 3 and 8 weeks, when most organs form
7
Q
What happens during the fetal period?
A
Growth and remodelling takes place
8
Q
When is the CNS susceptible?
A
CNS is constantly forming and remains susceptible throughout the development
9
Q
Whats the difference between cogential defects and birth defects and embryo loss?
A
Congenital defects- present at birth
Birth defects and embryo less- have intrinsic and extrinsic causes
10
Q
When is the heart sensitive to environmental teratogens?
A
3-6 weeks major congenital anomalies
7-8 weeks functional defects and minor anomalies
11
Q
When are Ears sensitive to environmental teratogens?
A
4-9 weeks major congenital anomalies
9-16 weeks functional defects and minor anomalies
12
Q
What are the symptoms of fetal alcohol syndrome (FAS)?
A
Characterised by small head size, narrow upper lip and low nose bridge
Brain can be dramatically smaller (defects in neuronal and glial cell migration, abnormal cell death)
13
Q
How frequent is FAS?
A
FAS is the 3rd most prominent cause of neurological problems and affects 1 of 500-750 births in USA
14
Q
How does FAD (fetal alcohol disease) impact mice?
A
Failure of the anterior neural tube to close results in anencephaly (baby is born without parts of the brain and skull)
Abnormal nose or upper lip (also seen in human)
3-D recontruction of MRI of mouse brain shows brain impacts olfactory bulbs and cerebral hemispheres
Cell adhesion mediated by L1 is inhibited by alcohol
15
Q
How does FAD impact brain development?
A
Nile blue stains show that embryos exposed to alcohol show more cell death compared to control
16
Q
How can drugs affect cell metabolism or cell proliferation impacts embryos?
A
This can cause limb defects in experimental animals, if administered during period of limb growth (4-12 weeks in humans)
17
Q
What was the case study for Thalidomide?
A
It is a painkiller for pregnant women in the 60s resulted in 5000 live births of affected children. It impacts limb development eg Phocomelia
18
Q
What impacts the defect through drug exposure?
A
Amount ingested, susceptibility of individuals and the period of development during and which drug was present
19
Q
What are the 3 types of congenital limb defects?
A
Reduction defects - part of entire limb missing
Duplication defects- Polydactyly
Dysplasia- Malformation eg fused digits, excessive growth of limbs or parts of limbs
20
Q
What can cause defects during development?
A
Compodents dont form during development, primorida fail to differntiate, duplication, overgrowth or undergrowth (hyper- or hypoplasia) focal defects and general skeletal abnormalilites
21
Q
What is the known gene that causes each of the defects?
A
HOXD13 = Syndactyl (fusion of digits)
FGF receptors 1 and 2 = Syndactyl plus malformations of the skul (Apert and Pfeiffer syndrome)
FGF receptor 3 = Dwarfism and parts of limb lost
TBX5 = Holt-Oram syndrome, heart defects, meromelia and finger thumb
22
Q
What are the symptoms of Apert syndrome?
A
Tower skull and syndactylyl
23
Q
What causes Apert Syndrome?
A
A gain of function mutation in the receptor which is hypersensitive and can respond to additional ligands. This results in premature differentiation, fusion of sutures in the skull and death of carilage om the growth plates of long bones
24
Q
What is SRY?
A
Putative transcription factor expressed in male genital ridge
25
What is the outcome with SRY transgenics?
Creates XY females and XX males
26
What is the method for 'conventional' transgenic mice by pronuclear injection?
Injection of DNA into the male pronucleus of mouse egg, transfer into foster mouse
Random insertion of DNA results in a 'transgenic' embryo, usually expressing multiple copies
Detected by Southern blot or PCR in tail biopsy
Copy number and integration site leads to variable phenotypes and mutiple lines have to be analysed
This approach can also be used to inject sgRNA with CRISPR/Cas9
27
What happens with neural tube defects?
Failure of part of the neural tube to close disrupts differentiation of CNS; the indunction vertebral arches and causes a number of developmental anomalies (mild to fatal)
28
What can prevent neural tube defects?
Folic acid (Vitamin B12)
29
What are the potential neural tube defects?
Open cranial or caudal neuropore
Open vertebral canal = spina bifida
30
What are the 4 main methods for identiying genetic causes of birth defects?
Positional gene cloning
Mutagenesis screens in animal models (cloning of human homologues)
GWAS- genome wide association studies
Exome sequencing (examine 'Trio' of paresnts and affected child)
31
What is positional gene cloning?
Linkage (pedigree) analysis highlights a region of the genome where a mutant gene may riside
This region differs between people who have the condition and people who dont
32
What is the PAX6 cas study?
PAX6 transcripts are expressed in the retina and presumptive iris during fetal development
Pax6 knockout mice have a small eye phenotype
One copy of the Pax6 gene is either lacking or mutated in patients with Aniridia
In Drosophila LOF mutation of the homologous gene, eyeless, results in lack of eyes
Targeted overexpression leads od ectopic eye formation
33
What is a candidate gene?
Gene that is believed to be related to a particular trait, such as a disease or a physical attribute. Often a similarity between phenotype of knock out mice with a human syndrome
34
What is Waardenburg syndrome type 2?
Autosomal dominant loctaed on the short arm of chromosome 3
Symptoms: Deafness, multicoloured irises and white forelock
35
What conditon in mice is similar to Waardenburg syndome?
Microphthalmia "small eye"
Mutations in Mitf cause deafness, white patch of fur and eye abnormalities
36
What did the similarities between Waardenburg syndrome and Microphtalmis mean for candidate genes?
Human MITF became a candidate gene for Waardenburg it mapped to the correct region on chromosome 3 and patients has a mutation in the MITF gene
37
What are haploinsufficiency?
The phenotype occurs when only one allele is functional
38
What is pleiotrophic?
Pleiotrophic genes impact a number of tissues/organs
39
What are the symptoms of Piebaldism?
Sterility, anemia, pigmentation changes in skin and hair, defective development of gut neurons and the ear
40
What causes Piebaldism?
Mutation from the KIT gene, which is essential for the proliferation and migration of neural crest cells, germ cell and blood cell precursors
41
What is genetic heterogeneity?
Different mutations can lead to the same phenotype
42
How does genetic heterogeneity occur?
If the genes act in the same pathway a mutation in any of them can give a similar result - either the same or overlapping phenotype
43
What is an example of genetic heterogeneity?
Waardenburg syndorme (MITF) vs Piebaldism (KIT)
Sonic hedgehog (SHH) vs Holoposcencephaly
44
What is SHH mutation?
A knock out of SHH gene have cyclopia
45
How does the SHH gene work?
Activation of the pathways is caused by the Hh-ligand binding to patched-receptor relieves the inhibition of smoothened (smo) and blocking the cleavage of Ci/Gli
Full length Ci/Gli is a transcriptional activator, proteolytically cleaved Ci/Gli is a repressor
46
What are the symptoms of the SHH gen mutation?
Deletions in the human homologue of Shh result in mid-facial defects = holoprosencephaly (recessive)
Also seen in fetal alcohol syndrome - variable phenotype, mild to severe eg abscence of nose and cyclopia
This phenotype is mimicked by Shh null mice (knock out) or by inhibitors of cholesterol
47
What is the consequence of inhibiting cholesterol biosynthesis?
Holoproscencephaly
48
What happened to lambs or calfs when exposed to plant alkaloids (teratogen)?
Exposure can cause the development of cyclopia
49
What Smith-Lemli Opitz syndrome?
Mutant enzyme in cholesterol pathway
50
How frequent is Smith-Lemli Opitz syndrome?
1/9000 biths caused by autosomal recessive
51
How are loxP sites generated?
An allele is flanked by loxP sits are generated by homologous recombination- so called "floxed"
loP sequences are recognised by the cre-recombinase
If sites are in tandem the flanked DNA is excised
52
What happens to the allele flanked loxP sites?
Floxed mice are crossed with a mouse that expresses the cre-recombinase in a tissue specific manner
The recombinase will bind to the loxP sequences and excise DNA leading to KO
53
What is CreERT2?
A modified version of the cre-recombinase which needs to be introduced by tamoxifen (an estrogen analogue)
54
What is the advantage of CreERT2?
This provides an additional level of control: the time of deletion can be controlled through administration of the drug
55
What is ALS/Motorneuron disease?
Progressive muscle atrophy due to neuron degeneration and lack of innervation
56
How can you combat detrimental effects of ALS?
Muscle upregulations a microRNA (miR-206) to combat detrimental effects
57
What is the role of miR-206?
miR-206 downregulates HDAC4, which leads to production of a growth factor that stimulates nerve regrowth
58
What is miragen?
The possibility of using microRNA mimics as therapeutics
For some diseases it may be be possible to use microRNAs as therapeutics by using antisense inhibitors
59
What is Marfan syndrome (MFS)?
Mutations in fibrillin-1 gene and connective tissue defects
60
What are the symptoms of MFS?
High risk of aortic aneurism and death due to internal bleeding, myopia due to lens dislocation, tall stature, hypermobile joint, scoliosis
61
What does MFS do biochemically?
MFS correlates with increased Tgf-beta
62
What happens with Tgf-beta neutralising?
Inhibition of Tgf-beta using neutralising antibodies (NAb) reduces disease features
63
How can Losartan be used as a treatment for Tgf-beta?
Losartan is an antagonist of Tgf-b signalling
64
What was the outcome for using Tgf-beta as a treatment for MFS?
Reduces MFS phenotype to almost normal when given to pregnant mice
Losartan led to a better outcome compared to Propoanolol ( a beta-blocker used in hypertension)
65
What is the definition of cloning?
Making an exact copy of an original form, derived form klon = greek for twin or breaking in two
66
When does cloning happen in life sciences?
Term originally reffered to organisms that use asexual reproduction eg a bacterial clone
67
When does cloning happen in molecular biology?
'cloned genes' are placed into bacteria to be repliocated
68
When does cloning happen in science fiction?
Clones are multiple copies of an original, eg Star wars 'War of the Clones'
69
When was the first asexually produced sheep?
1996
70
What was involved in reproducing the sheep asexually?
Using diploid, somatic cell and enucleated egg. The asexually produced organism had the same nuclear genome as the donar of the somatic cell
71
What are somatic cell?
Somatic (cells) contain two sets of chromosomes. In higher eukaryotes they are dipliod
72
What creates haploid cells?
Meiosis creates haploid germ cells
73
What happens in sexual reproduction?
Fusion of two haploid germ cells (male and female), restoring diploid number
74
What are the advantages of sexual reproduction?
Many unique combinations of maternal and paternal chromosomes
Crossing over= genetic exchange between homologous chromosomes
75
What does cell fusion show about differentiation programmes?
That differentiation programmes are reversible, including gene inactivation
76
How does cell fusion show that differentiation programmes are reversible?
Exposure of a human liver cell nucleus to mouse muscle cytoplasm leads to activation of human muscle-specfic genes and repression of liver-specific genes
77
How can differentiated nucleus be reprogrammed?
Genes that are not expressed can become reactivated in the right circumstance
78
What is totipotency?
A single nucleus contains all the information to generate a whole genome
79
What are lower eukaryotes diploid and haploid stage?
Haploid is the living stage with sexual reproduction producing diploid cells. These then undergo meiosis to produce haploid cells.
80
What was the most dramatic experiments to show how reversible is cell differentiation?
In the 1950s, the most dramatic experiment had the nuclei from cell at different stages of development to replace the nucleus of an egg.
If the nucleus can support and drive development this would indicate that no irreversible change had occured to the genome during differentiation.
81
How was the first experiments of adding cell nucleus into an egg?
They were first carried out in amphibians, which have large eggs that are very robust and accessible. The genetic material in the egg's nucleus is destroyed by UV irradiation and a nucleus from a differentiated cell is microinjected
82
What were the types of DNA that was added to the eggs?
Nuclei were taken from an early developmental stage, the blastula as well as adult DNA
83
Which nuclei of tadpole cells could support embyronic developement?
Adult skin, kidney, lung or tadpole intestinal or skeletal muscle cells
84
What happens when the nuclei of the those cell types interacted with cytoplasmic factors?
They behave as the genes in a newly fertilised egg's nucleus would
85
What were the results of John Gurdon's serial transplantation in Xenopus leavis?
That the egg cytoplasm is important in reprogramming and rejuvenating nuclei
86
How did John Gurdon discover the importance of egg cytoplasm in reprogramming nuclei?
He took adult skin cells to fertilise enucleated eggs, let them develop into a blastula, then dissociated the cells to obtain another nucleus which was placed into another egg. This increased the number of tadpoles that would develop
87
Why can cells from different developmental stages be reprogrammed to act like newly fertilised egg?
Nuclei form different developmental stages and different types of somatic cells are genetically equivalent and contain the complete genome (with the exception of permenant rearrangements of DNA during recombination in B and T-lymphocytes)
88
What was the time difference between John Gurdon experimenting on cloning Amphibians and cloning mammals?
Somatic cell nuclear transfer in amphibians took place in the 50s and 60s and it took 40 years before equivalent procedure took place in mammals primarily sheep and mice
89
When was Dolly the sheep?
1996 which was the first mammal cloned form adult cells
90
How were mice cloned?
From adult somatic cells and form embryonic stem cells
91
What increases success rate of cloning mammals?
Success increases significantly when developmentally younger cells are used
92
Why did the popular press became interested?
The popular press became interested, because Dolly is genetically identical to the sheep from which the somatic cell was taken. In other words the two are genetic clones
93
Why arent Dolly and the donor not truely identical?
Genes are not all that determines the phenotype of an organism, there are epigenetic or environmental influences, which are very important too, and therefore it is not possible to create truly identical individuals
94
What are the issues with cloning?
Important ethical and legal issues have to be addressed now since this technology may allow one day the creation of human individuals that carry the same genetic information as a previously existing individual
95
What is the basic recipe for cloning?
1 somatic (diploid) donor cell
1 unfertilised recipent egg
1 electric pulse
96
What needs to be prepared for the somatic donor cell?
Place in culture, serum starve to remove growth factors and cuase cell cycle arrest and induce 'quiescent' state
97
What needs to be prepared for the unfertilised recipent egg?
Its haploid nucleus removed or the genetic material destroyed (enucleated)
98
Why is an electric pulse needed for cloning?
To fuse cell membranes, simulate fertilisation and activate cell division (cleavage)
99
What is the membrane mV, how does it change and whats its function?
The resting membrane potential is minus 70 mV and this rapidly changes to plus 20 mV and then slowly returns to the original value. This mechanism may provide a fast block to polyspermy.
100
What are the uses of cloning?
Potential of cloned livestock
Animal models of disease
Clone rare animals to save endangered species
Cloning of your deceased pet?
Cloning of valuble race horse?
101
What are the uses of cloned livestock?
Replication of transgenic animals, engineered to produced a desired product for example milk
Human therapeutic proteins (hormones, insulin, clotting factors)
Organs for transplants (xenografts)
102
What mammals have been cloned?
Mice, sheep, cat, cow, deer, mule, horse, rat, wolf and more
103
Whats the disadvantages of cloning?
Cloning is still very inefficient and fundamental mechanisms are poorly understood
104
What is an example of animals not being completely identical due to epigenetic factors?
Pigmentation in calico cats is dependant on X-chromosome inactivation
105
What was the first non human primate cloned?
A rhesus monkey
106
What faciliate dteh cloning of the rhesus monkey?
Exposure of the nuclei to a demethylase enzme and a dug that inhibits histone deacetyalyes (Trischostatin A)
107
What happens during normal fertilisation?
Normal development requires contributions from both paternal and maternal genomes
Paternal and maternal genomes are modified or imprinted, during germ cell differentiation
Studies in mice have shown that certain genes in eggs and sperm are switched off during development by a process called imprinting.
Maternal and the paternal genome make different contributions to the development of an embryo.
108
What is the disadvantage of using nuclear transplantation?
Nuclear transplantation was used to generate mouse eggs which have either two paternal or two maternal genomes. The resulting embryos are known as androgenetic and gynogenetic. Both types have a diploid number of chromosomes, but their development is abnormal
109
What are the consequences of using two maternal genomes?
Embryos that develop from an egg with two maternal genomes have underdeveloped extraembryonic structures, such as placenta and yolk sac. This results in development being blocked, although the embryo itself is relatively normal and well developed.
110
What are the consequences of using two paternal genomes?
Embryos that develop from an egg with two paternal genomes have normal extra-embryonic structures, but the embryos itself only develops to a stage where a few somites have formed (3 weeks in human).
111
Why cant mammals be produced by parthenogenectically?
This clearly showed that both parental genomes are required for development and that they function differently and each make distinct contributions to the early embryo. This is the reason that mammals cannot be produced parthenogenetically, by activation of an unfertilized egg.
112
What causes hydatidiform mole?
Molar pregnancies are uncommon and occur when there is fertilization of an ovum by a sperm but loss of maternal chromosomes, leaving a 46XX karyotype composed only of paternal chromosomes, enough to form a placenta, but not a fetus. The result is a mass of tissue with grape-like swollen villi.
113
What is a dydatidiform mole?
A hydatidiform mole is an abnormal fertilized egg. The abnormal egg develops into a hydatidiform mole rather than a fetus (a condition called molar pregnancy).
114
When can a dydatidiform mole develop?
A hydatidiform mole can develop from cells that remain in the uterus after a miscarriage or a full-term pregnancy. Rarely, a hydatidiform mole develops when the fetus is normal
115
What is the overview of the types of hydatidiform mole?
About 80% of hydatidiform moles are not cancerous and disappear spontaneously
80% of hydatidiform moles are not cancerous and disappear spontaneous
Of these invasive moles, 2 to 3% become cancerous and spread throughout the body; they are then called choriocarcinomas
116
How can choriocarcinmas spread?
They can spread quickily through lymphatic vessels or bloodstream
117
What increases the risk of hydatidiform mole?
The risk of hydatidiform moles is highest for women who become pregnant before age 17 or in their late 30s or later.
Hydatidiform moles occur in about 1 of 2,000 pregnancies in the United States and, for unknown reasons, are nearly 10 times more common among Asian women.
118
What is imprinting and how many genes are imprinted in mice DNA?
Imprinting regulates gene activity, imprinted genes are usually inactive with Imprinting correlating with methylation.
A few hundred genes are imprinted in mice
119
What is an example of an impronted gene?
Imprinted genes are involved in growth control, for example insulin-like growth factor 2 (Igf-2)
120
What can be caused by imprinting?
Some of the developmental problems of cloned embryos are linked to imprinting
121
What does impriniting a gene impact and how did we discover this?
Imprinted gene not only affect early development but also the later growth of the embyro. Evidence for this comes from chimeric embryos generated between normal embryos and gynogenetic or androgenetic embryos.
122
What was the difference between normal mouse balsula with gynogenetic DNA and normal mouse balsula with androgenetic DNA?
When the inner cell mass from gynogenetic embryos is injected into a normal blastula growth is retarded (by up to 50%)
When the inner cell mass from androgenetic embyros is injected into a normal mouse blastula growth is increased up to 50% in the resulting chimeric embyros
123
What do they believe is the cause between the difference in normal mouse balsula with gynogenetic DNA and normal mouse balsula with androgenetic DNA?
This excessive or retarded growth may in part be due to the funciton of the Igf-2 gene which is imprinted in the maternal genome and therefore turned ‘off’. Only the paternal gene is active
124
What evidence did they have on Igf-2 gene imprinting impacting embryo growth?
The Igf-2 gene is imprinted comes from experiments using sperm carrying a defective Igf-2 gene to fertilize a normal egg. The resulting offspring is smaller. This is because only very low levels of Igf-2 are produced from the maternal allele which cannot make up for the loss of Igf-2 expression from the paternal genome.
In contrast, if the non-functional mutant Igf-2 gene is carried by the eggs genome development is normal. The required amounts of Igf-2 are being produced by the paternal allele.
125
What is the use of therapeutic cloning?
Therapeutic cloning would generate compatible embryonic stem (ES) cells to replenish dying or diseased cells in a patient with a degenerative disease or injury.
126
What is the advantage of therapeutic cloning?
These ES cells would be identical to the donor and immune-rejection of grafted cells (maybe someday tissues or organs) could be avoided
127
What is the method for therapeutic cloning using inner mass cells of blastocyst?
The cloned embryo would be generated by somatic cell nuclear transfer using a cell from the patient and a donated egg.
The embryo would exist in culture until it has developed into a blastocyst (day 5, pre-implantation).
ES cells would be isolated from the inner cell mass of the blastocyst. These could then be differentiated in culture into the desired cell type that is needed in the patient.
Shown to have worked
128
What is the method for therapeutic cloning using isolated primordial germ cells from a fetus?
Registered human stem cell lines exist and could be used for therapy. Problem: incompatibility and immune rejection by the patient (host)
Shown to have worked
129
What is the method for therapeutic cloning using adult stem cells?
Obtain adult stem cells from the patient and grow in a manner that allows them to become pluripotent
This remains experimental
130
What is the method for therapeutic cloning using adult somatic cells
The nucleus of an adult cell (somatic,diploid) could be isolated and transferred into an enucleated oocyte, which will develop into a blastula from which pluripotent “embryonic” stem cells can then be isolated and grown.
Original report in science was discredited
131
How have they demonstrated the curing of genetic diseases in mice?
Rag2 deficient mice cannot recombine their DNA to generate anti-bodies.
A somatic nucleus from the tail was used to generate ES cells.
These were by homologous recombination, cultured, expanded, differentiated and then re-introduced into the mouse (patient).
Mature antibody producing cells were detectable within a month after transplantation
132
What is the function of adult stem cells?
In the adult organism stem cells are important for maintaining tissues (continued production of blood, hair, skin, intestinal epithelial cells in the colon or Hematopoietic stem cells (HSC) in adult bone marrow)
133
What are the disadvantages of using adult stem cells?
Stem cells are known to be present in most differentiated tissues but they are highly elusive, very few markers are known
Compared to ES cells adult stem cells are not as proliferative and grow more slowly
Adult stem cells have a more limited potential to give rise to other cells types compared to ES cells
134
What are the advantages of using adult stem cells?
If isolated from the patient directly they are immune-compatible
135
Where do adult stem cells live in mice?
These include highly differentiated tissues and organs, such as muscle, brain and dermis of the skin and the tail.
136
How do you generate induced pluripotent stem cells?
iPS cells were generated by re-programming factors previously identified in embryos
137
What are iPS cells?
iPS cells: adult somatic cells that acquire pluripotency through re-programming by embryonic pluripotency factors
138
What is used in maintaining pluripotent stem cells?
Many of the factors that are important for the maintenance of the pluripotent stem cell ‘state’ have been extensively studied they include Oct-4, Sox-2, myc, Klf4, nanog and others
139
Who won the nobel prize in 2012 fro Physiology and Medicine?
Shinya Yamanaka
140
What discovery gave Shinya Yamanaka the nobel prize
He studied genes that are important for stem cell function
He transferred four such genes into cells taken from the skin
These cells were reprogrammed into pluripotent stem cells that could develop into all cell types of an adult mouse
He named these cells induced pluripotent stem cells
141
What was the impact of Shinya Yamanaka's discovery?
iPS cells can now be generated from humans, including patients with disease. Mature cells include nerve, heart and liver cells thereby allowing scientists to study disease mechanisms in new ways
142
What happens to the stem cell factors during ES cells differentiation?
They get switched off
143
What happens if ES cells are not fully differentiated?
If cells are not fully differentiated they form teratomas as shown after transplantation into immunodeficient mice
144
What is the next step for ES cell cloning therapies?
Direct trans-differentiation of somatic cells
145
What discovery gave Shinya Yamanaka the nobel prize
He studied genes that are important for stem cell function
He transferred four such genes into cells taken from the skin
These cells were reprogrammed into pluripotent stem cells that could develop into all cell types of an adult mouse
He named these cells induced pluripotent stem cell
