genomic imprinting

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  • Created by: Sarah
  • Created on: 23-05-19 16:20
what is genomic imprinting?
parental-specific gene expression in diploid cells
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where is the gene expressed from?
only from maternal or paternal chromosome
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what was known about mutations being inherited 45 years ago?
that some mutations had different impacts on the development if they are coming from a father or mother
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what is an example of this?
mutation in mice- if the heterozygous male is crossed with a normal female the mutation is inherited from the paternal genome then the progeny of the mouse is normal, viable short tail
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what happens if the heterozygous female is crossed with a normal male?
the mutant copy of the gene is coming from the mother then its lethal, has severe effects- overweight, short tail, polydactyl
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what did this lead to the idea of?
that some genes are expressed only from the maternal or paternal chromosome = genomic imprinting
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what is genomic imprinting?
1 copy of gene from mother and 1 copy from father, genomic imprinting- certain genes are expressed in parent of origin specific manner = a gene from mother or father is epigenetically silenced
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What does it mean that some genes are expressed from either maternal or paternal copy of genes?
this means both genomes are needed for reproduction and healthy development
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what happens with gynogenetic embryos (where 2 maternal genomes are implanted into oocytes)?
lethal during embryogenesis - defects in extraembryonic tissues
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what happens with androgenetic embryos where the nucleus from the oocyte was removed and 2 paternal haploid nuclei was put into the oocyte?
they die early in development but due to defects in embryonic tissues
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what are these extraembryonic (gynogenetic) or embryonic (androgenetic) thought to be caused by?
a dominant effect of a few imprinted genes
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how many imprinted genes are there?
150 imprinted genes- genes that are expressed either from the maternal or paternal chromosome
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what do these imprinted genes form in the genome?
80% of these genes are clustered forming separate clusters in the genome and 80% are clustered in 16 genomic regions that contain 2 or more genes
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how many clusters are well characterised?
7 clusters well characteristised- contain 3-12 imprinted genes spread over 82-3000 kbps of DNA
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what is genomic imprinting heavily reliant on?
DNA methylation = silencing one of the parent genes
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What was the only modification shown to function as a genetic imprint?
DNA methylation (diff to others histone mods and modifying enzymes small role only relies on DNA methylation here most things usually histone mod and DNA methylation but only DNA meth here)
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what is a differentially DNA-methylated region (DMR)?
DNA sequence carrying a gametic methylation imprint.
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is there more paternal or maternal methylated DMRs known?
many more materanlly methylated DMRs (differentially methylated regions)
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what is an imprint control element (ICE) for?
region which controls the expression of the whole or part of an imprinted cluster
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in 7 well characterised clusters what is the imprint control element?
the differentially methylated region
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what happens if you delete the imprint control region?
causes loss of imprinted expression only when deleted from the parental allele expressing the long non-coding RNAs
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where are protein coding genes and long non coding RNAs expressed from?
opposite parental chromosomes
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what is the range in cluster size?
from 80kbps to 37,000 kbps
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what is the range in gene number in these clusters?
3 to 13 genes
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how is it complementary maternal and paternal chromosomes?
most of the clusters there is protein coding genes expressed from one chromosome either maternal or paternal and there is non coding RNA that is expressed from the other chromosome either maternal or paternal
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when are imprints aquired?
in the gametes when maternal and paternal genomes are separate
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why does it make sense that the imprints are acquired when maternal and paternal genomes are still separated?
as when they come together in the nucleus there's no way the nucleus will know which ones from the mother and which ones from the father
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is there any sequence similarity between known DMR sequences?
no no sequence similarity between them so there's no specific motifs that indicate this is maternal or paternal genome is recognised as methylated
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what does the DMRs have several common features to?
several common features that these DMRs consit of tandem repeats that may adopt secondary structures that attract enzymes and induce DNA methylation
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we know more about maternally methylated DMRs what do these contain?
CpG rich pairs spaced by 8-10 bps
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why is 8-10bps between CpG rich pairs in maternally methylated DMRs ideal
spacing is optimal for recognition by at least 2 methyl transferases-DMTM3A and DMT3;
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what different methyl transferases recognise these CpG rich sequences in DMRs?
DMT3L and DMTM3A
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what is the hypothesis about this CpG spaced marks?
transcription acorss DMR means this region has more open chromatin so hypothesis that this open chromatin domain together with specific CpG marjed space are permissive for DNA methylation
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what is maternal methylation of DMRs de novo promoted by?
transcription across the DMR
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What was concluded about this?
a combination of at least several factors provide a starting point for DNA methylation on DMR regions either in maternal or paternal genome when they're still separated
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what happens to this mark once its acquired?
its stably inherited across development, inherited through cell division but also escaped genome wide reprogramming after fertilisation
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what do DMR marks escape?
genome wide changes in methylation where methylation marks are erased and reset early in mammalian development
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when will the methyaltion marks need to be erased?
in primordial germ cells as in germ cells material is from maternal and paternal chromosomes so it would be a combination of both but depending on the gender of the animal it will need to erase and put methylation marks in the sex specific gamete
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3 steps in imprints in mammalian development?
1) acquisition- in gametes when maternal and paternal genomes separated 2) maintenance- stably inherited in cell division, escape genome wide reprogramming 3) erased- in PGCs to set new imprints depending on sex
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what 2 clusters are silenced by genetic imprinting?
Igf2 cluster and Igfr2 cluster
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what are the 2 types of cis-acting silencing of imprinted genes?
1) insulator model (Igfr2 cluster) 2) long non-coding RNA mediated silencing (Igfr2 cluster)
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what is cis-acitng?
acts on the same DNA strand
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what is the insulator model based on?
Igf2 cluster
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what is the other cluster?
Igrfr2- long non-coding RNA mediated silencing
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what is IGF signalling important for?
development, growth control, metabolism control and differentation
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what happens when IGF signalling is dysregulated?
promotion of diseases like cancer
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what does IGF stand for?
insulin like GF
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what are the 2 different receptors for IGF signalling?
IGF1 and 2 receptor
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what does the IGF 1 receptor do when its activatyed?
promotes growth and differentiation when its activated by IGF1
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what can IGF 2 bind to?
IGF2 can also bind both IGFR 1+ 2 receptor
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what's IGF 2 very important for?
correct growth and differentiation
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what gene does the IGF2 cluster contain?
the IGF2 gene
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what happens to the IGF2 gene in the IGF2 cluster?
the genome is silenced in a stress specific manner
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what is this model an example of?
science is not a large breakthrough but one step at a time that brings final knowldge
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what did they find binds unmethylated ICes (imprint control elements)?
CTEF
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what is CTCF?
a zinc finger protein
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what was CTCF already implicated in?
chromatin boundary formation
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what did the authors find here about CTCF?
it also binds to insulation imprinting elements only when they're unmethylated
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what experiement did they do to show CTCF (zinc finger containing) binds to unmethylated imprint control elements?
DNA probes of the imprint element were either unmethylated or methylated in vitro in a tube and incubated with recombiannt CTCF protein- see if CTCF can bind DNA region after its pulled down and put on gel and visualised if CTCF present
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what would be found if CTCF can't bind the DNA?
precipitation wouldn't result in CTCF being pulled down so there'd be no protein int he reaction and there would be no signal on the western blot
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what did they find?
CTCF is only present (binding) when the DNA probe of different imprint elements were unmethylated
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what was it shown about a region in the IGF2 cluster?
there's a region which works as a transcriptional insulator, 2 important genes in this cluster- IGF2 and RNA H19, also shows DMR region
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what does the H19 code for?
non coding RNA
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what do the enhancers do?
drive expression of both H19 and IGF2 in a tissue specific manner so one of them is specific for muscle cells and one for epithelial cells
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what did the authors create?
2 trangenic mice- similar but 1 had a deletion where DMR was so it lacked the DMR region, the other had an additional DMR inserted between the 2 enhancers
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so transgenic mice, one with no DMR and one with additional inserted DMR, then what did they look at?
how H19 and IGF2 are expressed depending on whether they inherit the mutant allele from the mother or father- used SNuPE technique to look for differential expression
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they used the SNuPE technique to look ofr differential expression what does this stand for?
single nucelotide primer extension
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what is the single nucelotide primer extension technique?
can be used for quantifying allele specific expression. Total RNA from a sample is located and find normal mRNA (polyA at 3') then using polyT primer and RT this RNA converted to cDNA- product of gene amplified using primers in PCR
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what is single nucleotide primer extension based on?
reverse transcription and PCR
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what happens after you've amplified the gene product (cDNA) with PCR?
products are leuted from a gel and they're separated into 2 different batches
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what is important here about the 2 different alleles of the gene?
the 2 different alleles of this gene has a single point nucleotide polymorphism so differ in a single nucleotide and this can be silent so doens't change aa but this can be used to quantify expression of 1 allele or the other
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where does the specific primer end up?
just 3' of this changed nucleotide are added and anneal to these strands in both batches but then instead of adding the DNTPs different aas added dependending on the polymorphism- nucleotides are labelled by radioactive phos or fluroscence now
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what is happening when these nucleotides and polymerase is added?
one reaction where we added DCTP only in the cDNA that has G can be elongated by one nucleotide that is labelled. in the other where we added DTTP the other cDNA is elongated by one labelled nucleotide how much labelled depends on product start with
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how much new molecule labelled will depend on what?
how much product of this particular gene there was in the first place expressed from each copy either maternal or paternal
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what was this technique used for?
to quantify expression of these 2 transgenic mice
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what did they find about the inheritance of H19 non coding RNA?
it is only expressed when it's inherited from the maternal allele
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when is IGF2 in normal mice expressed?
only when its expressed from the paternal allele
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what happened when DMR was removed?
both H19 and IGF2 were expressed from both maternal and paternal allele
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what did IGF2 and H19 being expressed from both maternal and paternal chromosomes when DMR was removed fit with?
which fit with the model that DNA methylation of DMR is required for imprinting and strand specific expression
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what happened when DMR was introduced between 2 different enhancers fo IGF2 and H19?
only expression of H19 either in the liver which is driven by this promoter or in muscle by a different promoter
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in the control where is H19 expressed when its inherited from the maternal allele?
in the liver and muscle
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what happened when there was the DMR inserted between the geens?
expression in the liver was normal but there was no expression from the muscle promoter which was separated from H19 by DMR
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what did this show?
DMR works as an insulator so it insulates the gene from the action of the enhancer and stops it being turned on
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insulator model of IGF2 cluster silencing?
paternal copy is methylated, methylation propagates into H19 encoding gene which silences it. Prevents expression of H19 from this allele and binding of CTCF
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What happens to the expression of IGF2 genes and other genes in this cluster in the paternal one when H19 is silenced?
enhancers can drive expression of IGF2 genes and other genes in this cluster which result sin the paternal copy expressing IGF2 and other protein coding genes but NOT H19
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what happens on the maternal chromosome in IGF2 mediated silencing?
opposite. DMR is not methylated which allows binding of CTCF which forms boundaries between diff chromatin domains and allows DMR to work as an insulator prevents enhancers acting on the the protein coding genes so they're not expressed
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why are the protein coding genes not expressed?
they can't sense the enhancers because of the insulator region DMR
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what is expressed from the maternal copy?
H19 long non coding RNA is expressed as its not methylated
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what is Beckwith-Wieldemann syndrome?
multiple developmental defects and depending on various factors babies survive and are born but have dev and other defects including microsomia, microcephaly, abdominal wall defects, neonatal hypoglycemia
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what can hypoglycemia lead to if left untreated?
peremant brain damage
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what causes Beckwith-Wiedmann syndrome?
uniparental disomy of IGF2 cluster- when maternal copy of the gene is replaced by an extra paternal copy- leads to too much IGF2 production
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why does it lead to too much IGF2 produciton?
both copies will express IGF2 (and other protein encoding genes)
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what else goes wrong in Beckwith Widemann syndrome?
IGF2 linked to differentation so defects are associated with processes where differentiation doesn't go right = cancer so patients have an increased chance in developing neoplasms in chuldhood
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what is the other method of silencing?
long non coding RNA-mediated silencing of the IGFR2 cluster
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how did they tell that the maternal DMR is methylated in the IGFR2 cluster?
HPA 2 hypersensitivity- hpa II can not cut where methylated?
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how did the experiment show it was the maternal DMR that was methylated in the IGFR2 cluster?
maternal locus is not cut- heavily methylated so not recognisable to Hpa II- can't cut, no difference in size when enzymes added = methylated
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model of IGFR2 cluster silencing?
maternal copy is methylated- prevents transcription because ICE overlaps with the promoter of long non-coding RNA so, if methylated pol 2 canNOT be recruited to this non-coding RNA and it won't be expressed but other pro coding genes like IGF2 will b
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if the ICE is not methylated then what can be expressed?
the non coding RNA
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what happens in the paternal copy when its not methylated?
non-coding RNA is expressed and mechanism not understood but it can propagate and prevent transcription of all other genes around by somehow inhibiting pol 2 (not undertsood how)
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what is the result of this differential methyaltion
on the maternal chromosome the protein coding genes are expressed and RNA not, on the paternal chromosome non coding RNA is expressed+ no pro coding genes = 1 chr RNA expressed, 1 chr proteins expressed
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some genes are expressed exclusively from either maternal or paternal chromosomes- what are these genes organised into?
clusters which usually contain at least one non-coding RNA
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are both maternal and paternal genotypes required for reproduction?
yes normally
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how were unimaternal progeny was produced?
embryonic stem cells obtained from female organisms and using cell culture tricks made haploid then used CRIPSR-KAS9 genome editing either 2 or 3 different imprinted loci deleted, modified cells (lack imprints)+normal ESCs injected enucleated oocytes
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where was the zygotes that formed implanted?
into host mothers
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3 locci deleted?
H19, Dlk1, Ragrf1
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what happened when 2 genomic imprints were deleted?
they all died early in PN development and had issues with movement, cholesterol + IGF1 levels
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what happened with deletion of 3 imprinted locu?
the uni-bimaternal mice were produced and behaviour was relatively normal- viable progeny size of litter normal. Bimaternal progeny efficiently produced but might not be the same for humans
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why was it more difficult to produce androgenic unipaternal mouse?
had to KO 6 of the 7 locii for genomic imprinted genes
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how id they make unipaternal from paternal chromosomes?
androgenic haploid genetic SCs were modified with KRISPR-KAS9 to delete 6 locci. They were then co-injected with sperm into enucleated oocytes- oocyte int host mother
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it didn't develop so what did they do?
used tricks to produce under androgenetic diploid ESCs from this one and implanted it into a tetraploid zygote which can't form embryonic structures so all embryonic structures made from androgenic diploid ESCs
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what happened to the pips which has KO of 6 imprinted loci?
defects in the eye structures, jaw structures- multiple developmental abnormalities and much higher birth and placental weight
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what happened when they removed 7 imprinted loci?
pups were born, looked normal, normal birth weight so they didn't have any obvious defects but died within the first 48hrs after birth no obvious defects tho
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where is the gene expressed from?

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only from maternal or paternal chromosome

Card 3

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what was known about mutations being inherited 45 years ago?

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Card 4

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what is an example of this?

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Card 5

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what happens if the heterozygous female is crossed with a normal male?

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