Call Girls Ahmedabad Just Call 9630942363 Top Class Call Girl Service Available
BIOL3200 PROPOSAL
1. INTRODUCTION
Mutant variants of the Prickle1 gene in humans have been shown to give rise to a previously
undocumented subtype of Progressive Myoclonus Epilepsy, a neurological disease characterized
by seizures and ataxia. Encoding proteins essential for polar cell planarity and transcription
regulation in neural cells, the Prickle1 gene is present as a homolog in the laboratory mouse.
Systematic genetic studies performed on laboratory mice can be used to investigate the gene’s
role in disease causation, withRNAi-mediated gene ablation and restoration coinciding with the
presence and absence of symptoms respectively. Cre-Lox technology was additionally employed
to maintain spatial and temporal disease manifestation.
HYPOTHESIS
The Prickle1 homolog in mice plays a significant role in differentiation, cell fate determination,
and cell signaling, with its ablation results in the development of a myoclonic epilepsy syndrome
similar to that seen in PME-positive humans. Restoration of gene function should likewise
alleviate symptoms. In vitro gene knockdown via RNA interference is analogous to the loss-of-
function observed in diseased individuals, and its functional ablation at different time points and
in different tissues should produce a pattern of characteristic symptoms that provide valuable
information about the disease.
AIMS:
- To investigate the effects of mutant Prickle1 proteins in mouse models such that more
can be elucidated regarding the syntenic human disease of Progressive Myoclonus
Epilepsy
- To investigate the severity and symptoms of the disease under spatial control
- To investigate the severity and symptoms of the disease under temporal control
BACKGROUND AND SIGNIFICANCE
A new subtype of Progressive Myoclonus Epilepsy (PME) has been documented in scientific
literature, with linkage analysis revealing no participation of known epilepsy genes. The disease
is familial, highly penetrant, and progressive, manifesting in childhood and characterized by
frequent, recurring seizures (synchronous spikes of abnormal brain activity). Ataxia, which is
dysfunctional muscle control, is generally observed and considered a hallmark sign, with patients
present with unstable gait and coordination difficulties. While a normal lifespan proves
achievable, a significant proportion of patients do not reach adulthood, due primarily to the
affected muscles impeding the essential processes of respiration and swallowing.
Displaying autosomal recessive inheritance patterns, the prevalence of PME is yet unknown,
though its high incidence in large, consanguineous Middle-Eastern families suggests a potential
founder effect caused by novel mutations in a single ancestral allele. Genetic analysis reveals a
2. transitional missense mutation in the Prickle1 gene mapped to human chromosome 15, with
corresponding orthologs found in Drosophila and Mice. The characteristic PET domain is largely
invariant between species, suggesting functional importance, and the common missense variant
R104Q observed within the PET domain of diseased individuals is thus thought responsible for
functional ablation and disease causation. Other Prickle1 variants are less commonly
documented, if at all.
The native Prickle1 gene performs a variety of functions, encoding five documented protein
isoforms. The 883 amino acid long Prickle1 protein is integral to the non-canonical Wnt
signaling pathway, participating in the biochemical cascade that, in conjunction with other
known signaling pathways, leads to planar cell polarity determination and associated neural cell
patterning. This includes neural tube closure and the regulated formation of the rudimentary
spinal cord, in addition to directed organ positioning with conventional asymmetry. The non-
viability of mice nullizygous for Prickle1 suggests its importance in early developmental
differentiation, though this embryonic lethality is not documented in human individuals.
Histopathological analyses of spontaneously aborted mouse embryos display chaotic cellular
positioning with skewed apical-basal orientation, with indistinct and improper segregation and
migration of the three germinal layers of the fledgling inner cell mass. The compromised
structural integrity is understandably fatal.
Prickle1 proteins are also suspected to work in coordination with the RE1-Silencing
Transcription Factor (REST), which is responsible for the transcriptional downregulation of
neuronal genes including those encoding neurotransmitters or receptors. Rigid regulation of
downstream genes is essential for the maintenance of coordinated neuromuscular function that
allows for the fine motor skills required in human kinesiology, and Prickle1 protein complexes
with REST, allosterically impeding its translocation to the cellular nucleus whereupon it can
constitutively suppress neuronal gene function. Conversely, ablation of Prickle1 protein leads to
ectopic and inappropriate REST-induced neuronal gene silencing, which may be responsible for
the ataxia/seizure phenotype observed in diseased individuals. Co-immunoprecipation has
revealed independent REST proteins in PME patients with a consequent downward shift on a
Western blot assay, suggesting that R104Q mutant Prickle1 protein is unable to bind to REST
and regulate its function.
Suspected secondary functions include encoding nuclear receptors specific for stereospecific
ligands, possibly with accessory zinc ions, and regulation of nuclear import, export, and
permeability.
Immunostaining with rabbit polyclonal antibodies revealed that Prickle1 proteins are expressed
within brain tissue, primarily within the hippocampus, thalamus, cerebellum, and cerebral cortex.
The expression is not extended to glia, though it is suggested that differential expression patterns
are concordant with developmental stage. The cerebellum’s indispensability for normal posturing
and balance strongly suggests that ataxia is the result of its abnormal function via mutant
3. Prickle1. Epilepsy syndromes have described dysfunctions in the other cerebral regions as
causative.
Elucidating the function of Prickle1 proteins and their behavior in both wildtype and mutated
forms can lead to the development of directed cures. As such, mouse models are employed for
investigation. Homozygous mutant mice are postulated to develop the same debilitating
symptoms as their human counterparts. Spatial expression of mutant genes allow for the
localization of affected tissues based on symptom quantification, and temporal expression of
mutant genes allows for the distinction of PME as either an inborn neurogenerative or an
acquired syndrome which may present only in later life.
The evolutionarily-conserved RNAi phenomenon is a highly-regulated form of endogenous,
post-transcriptional gene regulation. Processed, single-stranded RNA strands of intermediate
length (commonly 21-24 nucleotides) bind complementarily to nascent mRNA transcripts,
resulting in their systematic degradation via the catalytic components of the multiprotein RNS
Induced Silencing Complex (RISC) the former is incorporated within. The principle of antisense
RNA intervention can thus be replicated in vitro, facilitating the targeted knockdown of specific
genes. This abortive mechanism inhibits the downstream protein synthesis and processing,
effectively suppressing the gene’s inherent function.
Derived from DNA expression vectors, laboratory-designed shRNAs comprise antisense and
sense regions, making them recognizable substrates of the native murine DICER catalyst
responsible for their enzymatic introduction into a functional RISC complex. While concordant
with the delivery method, silencing rates of 90% have been documented, as quantified by
haplosufficiency assays.
While thus effective in gene ablation, in vitro RNAi is known for producing undesirable ectopic
effects. It is thus utilized in tandem with conventional Cre-Lox recombination technology that
provides a measure of spatial and temporal control.The bacterial-derived Crerecombinase protein
is capable of causing recombination events when exposed to DNA fragments bounded by
corresponding loxP sites. This recombination can be an inversion, translocation, or deletion,
depending on the orientation of the loxP sites, resulting in the formation of a stable Holliday
junction and the desired translocation event, after which endogenous DNA ligase catalyses
strand re-joining.
Thus, the introduction of specific mutations leading to disease development theoretically implies
that converse removal results in corresponding disease resolution. This hints at a potential cure,
though further scientific research is imperative before proper, significant conclusions can be
made.
EXPERIMENTAL DESIGN
Section 1– Introducing the R104Q mutation
4. Recessive diseases are generally characterized by loss-of-function mutations. However, it is
imperative to distinguish between homozygous mutants and the nullizygote. The viability of
human homozygotes indicates that Prickle1 function is still present, albeit at drastically reduced
levels. Nullizygote is therefore non-synonymous with homozygous mutant.
Fistly, we capitalize upon the RNAi pathway native to mice, in which experimentally-
designedshRNA hybridizes to complementary native sequences which are then systematically
degraded by the catalytic Argonaute action of the RISC complex the siRNAs are incorporated
into. Controlled by a proximal, ubiquitous UG promoter, the complementary nucleotide sequence
lies upstream of a Puromycin-resistance reporter. Mimicking murine microRNA, it is processed
by endogenous Dicer and Drosha enzymes and loaded into the cytoplasmic RISC complex.
Perfect complementarity results in enzymatic degradation of the immature Prickle1 transcript,
while imperfect complementarity leads to suppression of the RISC complex, reducing gene
expression in either scenario. This is particularly important, affording a measure of tolerance
against potential SNPs resulting from the degeneracy of the genetic code.
Figure 1.1.RNAi-mediated gene regulation/suppression
While commercial shRNAs can be bought via Origene, manual designing is possible so long as
the sequence of the desired knockout is known. shRNA presents a characteristic hairpin loop
5. structure achieved via self-complementation, and it is these sense and antisense regions that are
capable of hybridizing to the destined knockdown gene.
Figure 1.2. The characteristic shRNA construct used for in vitro gene knockdowns. Restriction
sites are required for introduction into recipient plasmids or viral vectors.
Figure 1.3.Prickle1-targetted shRNA (Origene), with a Puromycin resistant marker used in
selection for Lentiviral plasmids that have incorporated the construct via electrophoretic
transfection methods.
(Manual synthesis: Native U6 promoters contain essential GGG sites from which transcription is
initiated, and the Guanine repeats within the target gene sequence are thus considered suitable
start sites. Directly distal would be the selected sense sequence and the following reversed sense
site, with an intervening central 8-nucleotide loop containing a bacterial restriction enzyme site.
A second, complementary oligonucleotide sequence of similar configuration then hybridizes to
this strand, forming a double-stranded DNA fragment with overhangs specific to their internal
restriction enzyme recognition sites, the latter used to facilitate the fragment’s introduction into
viral or plasmid vectors.)
After integration of construct-carrying plasmids, Lentivirus-associated delivery was performed
due to its non-episomal nature. Unlike conventional Adenoviruses which do not integrate into
transcriptionally-relaxed euchromatin, Lentivirus show successful incorporation and thus
perpetuate through the progeny. Circumvention of random, potentially deleterious mutagenesis
6. was achieved by using an integrase-deficient lentivirus,Intranasal introduction was favoured as
an administration route as viruses show functional optimization in the lower temperatures of the
respiratory tract as opposed to the slightly-warmer temperature of blood.
Assuming sucessful gene knockdown, the mice should now exhibit the classical symptoms of
PME. 0.5cm of their tails was removed, and identification ofG(0) transgenic founder mice was
performed via qPCR with primers specific for the U6 promoter and Puromycin-resistant sites (5'-
CGAAGTTATCTAGAGTCGAC-3', and 5'-AAACAAGGCTTTTCTCCAAGG-3' respectively).
While mutant mice are expected to experience seizures, seizure incidence is unpredictable.
Injected administration of anxiogenicPentylenetetrazol is thus used to induce seizures in both
affected and control mice, with seizure susceptibility, intensity, duration, and recovery time
being documented. Simultaneous EEG monitoring is used to quantify abnormal brain activity,
while functional Magnetic Resonance Imaging can be employed to determine which cerebral
regions are active during seizure occurrence.
As ataxia is a hallmark symptom of PME, mice are subject to a battery of kinaesthetic tests. The
ability to balance on a horizontally-rotating rotarod and the presence/absence of tremors can
reveal functional disability in mouse motor coordination. Electronic gait analysis can be used to
document mouse walking stability and speed, both of which are believed to be compromised in
the ataxic mouse. General mouse behaviour is also documented, elucidating any secondary issues
that may be co-morbid with PME (e.g. below-average height/weight).
Section 2 – Conditional Mutagenesis (Cre-Lox Recombination)
RNAi usage circumvents embryonic lethality issues as shRNAs can be introduced at any age.
However, traditional Cre-Lox recombination provides an unrivalled measure of spatial and
temporal control. In the former, Cre is expressed only in a particular cell line, driven by a
specific promoter, and the corresponding translocation events will be exclusive to that cell line
when crossed with a mouse presenting specific LoxP junctions. In the latter, inducible agents
including tetracycline derivatives can be employed to catalyse time-specific translocation events
Thus, Cre-Lox mediated shRNA targeting the Prickle1 gene has been developed.
Here, a floxedStop cassette containing terminating thymidine repeatswas inserted within the U6
promoter driving shRNA expression such that RNAi performance is inhibited. The vector
construct was introduced via direct pronuclear injection, and crossing this floxed mouse with one
expressing Cre-recombinase would allow for the effective deletion of the intervening Neo
cassette, restoring U6 promoter function and the downstream RNAi-mediated silencing of the
Prickle1 gene product.
Crossing the floxed specimens with mice expressing Cre in different tissues allowed for the
elucidation of Prickle1’s role in the respective tissues.
7. 406-week-old C57BL6 mice were thus purchased from GENSAT.
- 10 of these mice were STOCK Tg(Dbp-cre)MN120Gsat/Mmucd (expression in Cerebral
Cortex, Thalamus, Cerebellum, Hippocampus)
- 10 of these mice were STOCK Tg(Ascl1-cre)ND216Gsat/Mmucd(expression in the
Hippocampus)
- 10 of these mice were STOCK Tg(Dlx5-cre)MO36Gsat/Mmucd (expression in the
Thalamus)
- 10 of these mice were STOCK Tg(Cxcl11-cre)KN257Gsat/Mmucd (expression in the
Cerebellum)
Figure 2.1. : A floxed STOP cassette introduced in a mouse prevents U6-driven shRNA
production. Removal via Cre restores this.
Floxed mice are then observed and subjected to the same behavioural tests mentioned in Section
1.1.
Gene ablation in different brain regions should lead to the manifestation of symptoms
corresponding to the region in question. Fluorescence signals are observed in respective brain
tissues under microscopy. 5 mice from each of the Cre-reporter lines are euthanized via cervical
dislocation, with transverse sections of brain tissue being excised and isolated. The sections are
homogenized and subjected to a classical Western blot procedure with protein presence and
molecular weight being quantified via polyacrylamide gel electrophoresis. This is compared with
a set of control mice.
Prickle1 protein is said to bind to the REST protein in vivo, and co-immunoprecipitation of
REST in mutant mice and controls will reveal the presence or absence of such allosteric
coordination.
Section 3- Inducible Cre-lox systems
8. The tetracycline-derivate of Doxycycline is used for the time-specific induction of Prickle1-
tagetted shRNA function. In this model, a tetR/O shRNA vector is employed, preventing the
constitutive expression of the shRNA that inactivates the Prickle1 gene via RNAi knockdown. In
the absence of the inducer, Doxycycline, the Tetracycline repressor binds constitutively to its
corresponding operator, disabling it. Both the repressor and the operator are part of the
insertional plasmid construct, the latter driven by a modified H1 promoter, with the former
controlled by a codon (CAG) one.
Figure 3.1: The introduction of Doxycycline-sensitive TetO and TetR elements can regulate
production of the shRNAs within the same construct.
Figure 3.2:Detailed manual synthesis of the inducible construct, requiring specific restriction
sites for incorporation into recipient plasmids/ vectors.
Doxycyline is introduced via intraperitoneal injection, whereupon it binds to the Tet repressor,
allosterically modifying its physical configuration such that a loss of necessary stereospecificity
is lost. This results in the physical inability to complex with the Tet operator just upstream of the
shRNA construct, restoring the former’s operative function. shRNAs are once more produced,
knocking down Prickle1 gene expression wherever expressed.
9. The mice are then observed and subjected to the same behavioural tests mentioned in Section 1.1.
Of interest to me is the investigation of PME as a developmental disorder or one that occurs in
the functioning adult brain. As Doxycycline injections allow for the temporal control over
symptom manifestation, different mice were given Doxycycline at different time points of their
life i.e. at 6-, 8-, 10-, 12- weeks of age and observed with regard to symptoms and behavioural
tests. Different degrees of affectedness would determine the disease classification i.e.
development of the disease within 6 weeks but not after that would indicate a
neurodevelopmental disorder.
HYPOTHESIZED RESULTS
Mice heterozygous for the Prickle1 mutation are likely asymptomatic, though haploinsufficiency
may be observed via protein quantification. Mice that undergo a loss of heterozygosity will
develop symptoms based on the cerebral region that the mutation was introduced in.
Table 4.1
Brain Region Symptoms observed
Hippocampus Seizures, with hippocampal sclerosis observed upon autopsy
Thalamus Generalized tonic-clonic seizures
Cerebral Cortex Focal or absence seizures
Cerebellum Ataxia
The same results are likely to be observed in floxed Mice (inducible or not).
Behavioral tests should show vastly different behaviour between mutants and controls.
Table 4.2.
Behavioral test Control Mice Prickle1-knockout mice
Rotarod Analysis Balancing ability intact Balancing ability compromised
Tremor Analysis No tremor observed Tremor of varying intensity
Tail Suspension Test Relaxed phenotype Clasping phenotype
Gait Analysis Predictable footprints Erratic footprints
Mouse Maze Performed quickly Performed slowly
It is worth noting that the discontinuation of Doxycycline treatment will possibly result in a
reversal of symptoms due to the impermanent nature of that inducible system. The restoration of
a normal phenotype may suggest that a complete cure is possible.
MATERIALS AND METHODS
10. Subjects: Healthy, 6 week old mice were obtained from the Jackson and GENSAT laboratories
and they were housed and euthanized according to guidelines meeting ethical criteria. An equal
number of male and female mice were obtained. Approximately half of the starter mice were
C57BL6 mice and the other half were Agouti, allowing for the identification of heterozygotes
and homozygotes via characteristic coat colour inheritance patterns.
Immunostaining protocols (Western Blots):
Blocking agent was 3% Bovine Serum Albumin. 0.5 micrograms/ml. Anti-prickle1 rabbit
polyclonal antibody was used. Secondary mouse anti-Prickle1 antibody was conjugated to a
fluorescent protein, producing a quantifiable signal which could be observed and analysed.
Protocol: Excised brain tissue segments were homogenized and treated with Sodium Dodecyl
Sulfate to confer upon them universal negative charges such that migration is determined only by
molecular weight. Samples are loaded into 5% Polyacrylamide gels and subjected to voltage
excitation in two dimensions, the first measuring isoelectric point, the other measuring molecular
weight.
The result is electroblotted onto Niitrocellulose membranes then probed with antibodies in a
sequential function. Mutant mice should show no or little Prickle1 protein product as compared
to control mice, which should show a Prickle1 protein profile similar to that documented from
known protein databases.
Polymerase Chain Reaction
Successful lentiviral-mediated construct uptake should reveal characteristic complementary
nucleotides upon PCR amplification. 5'-CGAAGTTATCTAGAGTCGAC-3', and 5'-
AAACAAGGCTTTTCTCCAAGG-3' primers were designed targeting the flanking U6 and
Puromycin sites, allowing for mass amplification of the shRNA construct (if present). The
presence of the construct would reveal a band (as made observable by treatment agent Ethidium
Bromide) upon subsequent polyacrylamide electrophoretic assaying; this band is not observed in
control mice.
Possible Limitations: RISC-oversaturation, Lentiviral infection, Lentiviral-degradation via
murine immunity, varying shRNA expression levels due to integration sites/copy number.
Reference list:
1. A Homozygous Mutation in Human PRICKLE1 Causes an Autosomal-Recessive
Progressive Myoclonus Epilepsy-Ataxia Syndrome, Alexander G. Bassuk,1,2,3 Robyn H.
Wallace,7 Aimee Buhr,
2. RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector,
VivekShukla, Xavier Coumoul#, Chu-Xia Deng, Int J BiolSci2007; 3(2):91-99.
doi:10.7150/ijbs.3.91
11. 3. Cre-lox-regulated conditional RNA interference from transgenes, Andrea Ventura,
Alexander Meissner, Christopher P. Dillon Department of Biology, Center for Cancer
Research, and Howard Hughes Medical Institute and §McGovern Institute,
Massachusetts Institute ofTechnology,
4. Conditional RNAi in miceAljoschaKleinhammeraJan Deussingc
5. A transgenic approach for RNA interference-based genetic screening in mice,
ShaohuaPeng , J. Philippe York, and Pumin Zhang
6. Taconic: Discover the Power of RNAiin vivo! Innovative RNAi gene knock down
technology for target validation,gene function analysis, and disease modeling.
THANK YOU FOR READING!!!