RNA interference (RNAi) is a mechanism where double-stranded RNA inhibits gene expression. It was discovered in plants, fungi, and animals. The mechanism involves dicer enzymes cleaving long double-stranded RNA into short interfering RNAs (siRNAs). These siRNAs are incorporated into an RNA-induced silencing complex (RISC) which guides the complex to mRNAs with complementary sequences, resulting in their degradation. RNAi has applications in therapeutics for cancer, viruses, and genetic disorders, as well as research in gene function and pathways.
RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA.
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA.
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
Most bacteria are free-living organisms that grow by increasing
in mass and then divide by binary fission.
Growth and division are controlled by genes, the expression
of which must be regulated appropriately. Genes
whose activity is controlled in response to the needs of a
cell or organism are called regulated genes. All organisms
also have a large number of genes whose products
are essential to the normal functioning of a growing and
dividing cell, no matter what the conditions are. These
genes are always active in growing cells and are known as
constitutive genes or housekeeping genes; examples include
genes that code for the enzymes needed for protein
synthesis and glucose metabolism. Note that all genes are
regulated on some level. If normal cell function is impaired
for some reason, the expression of all genes, including
constitutive genes, is reduced by regulatory
mechanisms. Thus, the distinction between regulated
and constitutive genes is somewhat arbitrary.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
Most bacteria are free-living organisms that grow by increasing
in mass and then divide by binary fission.
Growth and division are controlled by genes, the expression
of which must be regulated appropriately. Genes
whose activity is controlled in response to the needs of a
cell or organism are called regulated genes. All organisms
also have a large number of genes whose products
are essential to the normal functioning of a growing and
dividing cell, no matter what the conditions are. These
genes are always active in growing cells and are known as
constitutive genes or housekeeping genes; examples include
genes that code for the enzymes needed for protein
synthesis and glucose metabolism. Note that all genes are
regulated on some level. If normal cell function is impaired
for some reason, the expression of all genes, including
constitutive genes, is reduced by regulatory
mechanisms. Thus, the distinction between regulated
and constitutive genes is somewhat arbitrary.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes.
RNAi targets include RNA from viruses and transposons.
This is a presentation slide about cellular RNA interference process and RNA interference technology. Contains basic information about biology of cellular RNA interference processes and its discovery, and RNA interference technology. Also gives you the history and development of in-vitro and in-vivo technologies for applicability of RNA interference technology.
siRNA synthesis, siRNA libraries, siRNA delivering techniques, Electroporation, viral transfection methods, Advantages and disadvantages of RNA interference technology.
details about the preliminary and pre-clinical experiments of RNA interference as well as clinical trials of RNA interference.
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference.
RNAi is a powerful, conserved biological process through which the small, double-stranded RNAs specifically silence the expression of homologous genes, largely through degradation of their cognate mRNA.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
GLP applies to nonclinical studies conducted for the assessment of the safety or efficacy of chemicals (including pharmaceuticals).
GLP helps assure regulatory authorities that the data submitted are a true.
SMGT is a simple and efficient technique to produce transgenic mice
is a transgenic technique, uses the sperms as a natural vector to transport the internalize exogenous DNA into an oocyte during fertilization to produce genetically modified organisms.
Ribozymes (ribonucleic acid enzymes) are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
The Indian economy is classified into different sectors to simplify the analysis and understanding of economic activities. For Class 10, it's essential to grasp the sectors of the Indian economy, understand their characteristics, and recognize their importance. This guide will provide detailed notes on the Sectors of the Indian Economy Class 10, using specific long-tail keywords to enhance comprehension.
For more information, visit-www.vavaclasses.com
How to Split Bills in the Odoo 17 POS ModuleCeline George
Bills have a main role in point of sale procedure. It will help to track sales, handling payments and giving receipts to customers. Bill splitting also has an important role in POS. For example, If some friends come together for dinner and if they want to divide the bill then it is possible by POS bill splitting. This slide will show how to split bills in odoo 17 POS.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
Andreas Schleicher presents at the OECD webinar ‘Digital devices in schools: detrimental distraction or secret to success?’ on 27 May 2024. The presentation was based on findings from PISA 2022 results and the webinar helped launch the PISA in Focus ‘Managing screen time: How to protect and equip students against distraction’ https://www.oecd-ilibrary.org/education/managing-screen-time_7c225af4-en and the OECD Education Policy Perspective ‘Students, digital devices and success’ can be found here - https://oe.cd/il/5yV
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
2. Outlines
IntroductionIntroduction
RNA silencing
Definition of RNA interference
Discovery of RNAi
Mechanism of RNA interferenceMechanism of RNA interference
Applications of RNA interferenceApplications of RNA interference
Therapeutic applications
Other applications
3. RNA silencing
Several terms are used to described RNA silencing;
usually there are three phenotypically different but
mechanistically similar phenomena:
1. Cosuppression or post-trascriptional gene silencing
(PTGS) in plants
2. Quelling in fungi
3. RNA interference in animal kingdom
4. Definition
RNA interference (RNAi) is a mechanism that inhibits
gene expression at the stage of translation or by
hindering the transcription of specific genes.
RNAi targets include RNA from viruses and
transposons.
5. What is RNA interference (RNAi)?
“The Process by which dsRNA silences gene
expression...” Degradation of mRNA or translation
inhibition
6. Need for interference
Defense Mechanism
Defense against Infection by viruses, etc
As a defense mechanism to protect against transposons and
other insertional elements
Genome Wide Regulation
RNAi plays a role in regulating development and genome
maintenance.
30% of human genome regulated
7. Nobel prize winners in the C. elegans
field Sidney Brenner
John Sulston
Robert Horvitz
Andrew Fire
Craig Mello
8. RNAi was found to work in many
diverse species
Fungi
Trypanosomes
Insects
Zebrafish
Mice
10. In Interference
RNA
siRNA: dsRNA 21-22 nt.
miRNA: ssRNA 19-25nt. Encoded by non protein coding
genome
RISC:
RNA induced Silencing Complex, that cleaves mRNA
Enzymes
Dicer : produces 20-21 nt cleavages that initiate RNAi
Drosha : cleaves base hairpin in to form pre miRNA; which is
later processed by Dicer
11. siRNAs
Small interfering RNAs that have an integral role in the
phenomenon of RNA interference (RNAi), a form of post-
transcriptional gene silencing
RNAi: 21-25 nt fragments, which bind to the complementary
portion of the target mRNA and tag it for degradation
A single base pair difference between the siRNA template
and the target mRNA is enough to block the process.
Each strand of siRNA has:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’ overhangs
15. Difference between miRNA and siRNA
Function of both species is regulation of gene expression.
Difference is in where they originate.
siRNA originates with dsRNA.
siRNA is most commonly a response to foreign RNA (usually
viral) and is often 100% complementary to the target.
miRNA originates with ssRNA that forms a hairpin secondary
structure.
miRNA regulates post-transcriptional gene expression and is
often not 100% complementary to the target.
And also miRNA help to regulate gene expression, particularly
during induction of heterochromatin formation serves to
downregulate genes pre- transcriptionally (RNA induced
transcriptional silencing or RITSRITS)
16. Dicer
Loss of dicer→loss of silencing processing
in vitro
Dicer homologs exist in many organisms
including C.elegans, Drosphila, yeast and
humans (Dicer is a conserved protein)
RNase III-like dsRNA-specific ribonuclease
Enzyme involved in the initiation of RNAi.
It is able to digest dsRNA into uniformly
sized small RNAs (siRNA)
Dicer family proteins are ATP-
dependent nucleases.
Rnase III enzyme acts as a dimer
17. Dicer’s domains
1 4 32 2
Dicer is a ribonuclease (Rnase III family) with 4 distinct domainsDicer is a ribonuclease (Rnase III family) with 4 distinct domains:
1. Amino-terminal helicase domain
2. Dual Rnase III motifs in the carboxy terminal segment
3. dsRNA binding domain
4. PAZ domain (110-130 amino-acid domain present in protein like
Argo, Piwi..);it is thought to be important for protein-protein
interaction
18. RISC
RISC is a large (~500-kDa)
RNA-multiprotein complex,
which triggers mRNA
degradation in response to
siRNA
Unwinding of double-
stranded siRNA by ATP
independent helicase.
The active components of an
RISC are endonucleases
called argonaute proteins
which cleave the target
mRNA strand.
19. RNA i
( RNA Interference)
STEPSINVOLVED IN
RNA INTERFERENCE
20. RNA interference
RNA interference (RNAi) is a biological process in
which RNA molecules inhibit gene expression,
typically by causing the destruction of specific mRNA
molecules. Historically, it was known by other names,
including co-suppression, post transcriptional gene
silencing (PTGS), and quelling.
21. STEP 1
• dsRNA is processed
into sense and
antisense RNAs
• 21-25 nucleotides in
length
• have 2-3 nt 3’ overhanging
ends
• Done by Dicer (an RNase
III-type enzyme)
24. Step 4
RISC cuts the
mRNA approximately
in the middle of the
region paired with
the siRNA
The mRNA is
degraded further
25. What are sense and antisense
RNA?
Messenger RNA
(mRNA) is single-
stranded, called
"sense" because it
results in a gene
product (protein).
5´ C U U C A 3´ mRNA
3´ G A A G U 5´ Antisense RNA
26. What are sense and antisense
RNA?
Antisense
molecules
interact with
complementary
strands of
nucleic acids,
modifying
expression of
genes.
5´ C U U C A 3´ mRNA
3´ G A A G U 5´ Antisense RNA
29. Hematology (blood)
Hematologic disorders result from
Loss of gene function
Mutant gene function
Absent gene function
RNAi
May be used to create models of disease processes
Could help to develop pharmacologic and genetic therapeutic
targets
30. Oncology (cancer)
Targeting of oncogenes
Dominant mutant oncogenes, amplified oncogenes, viral
oncogenes
Define role of signaling molecules in tumor-creation
Improvement efficacy of chemotherapy and
radiotherapy
Tumor regression through creation of potentially new
mode of chemotherapy
31. Stem cell biology
Mouse research
Knock out tumor-suppression gene in mouse embryonic
stem cell
Observe tumor phenotype
Positive correlation between extent of Trp 53
(suppression gene) inhibition and severity of disease
32. Infectious Diseases
Virus targeting
RNAi – inhibit cellular and viral factors of disease
RNA transcriptase is RNAi target
Inhibition of replication
Main goal
Render cells resistant to infectious organisms
33. Hepatitis C
Infects ~200 million people worldwide
Often fatal
2002, Anton McCaffrey and Mark Kay at Stanford
University
Injected "naked" RNA strands into the tail veins of
mice
RNAi treatment controlled the virus in mice
34. Silencing genes in HIV
AIM:
Silence the main structural protein in the virus, p24,
and the human protein CD4.
Hit the virus where it counts by eliminating a protein it
needs to reproduce or cause infections.
35. Respiratory infections
RSV ( RESPIRATORY SYNCTIAL VIRUS), infects
almost every child by the age of two
Short strands of "naked" RNA
Controlled the virus in mice
Clinical trials are ongoing
36. Macular degeneration
Macular degeneration is the leading
cause of adult blindness
Excess VEGF which leads to
sprouting of excess blood vessels
behind the retina & obscuring vision.
The new RNAi drugs shut down
genes that produce VEGF. The drug
can be injected directly into the eye
First clinical trial: 24 patients,
launched in 2004.
Two months after being injected with
the drug, 6 of the patients had
significantly clearer vision
Other patients' vision had at least
stabilized
More extensive trials are ongoing
37. Huntington’s disease
Ideal candidate for RNAi therapy
Disease caused by protein, that
affects more than 30,000 people
in the U.S. alone.
We would want to shut down the
expression of the gene coding for
the abberant protein
2004, Beverly Davidson and
colleagues at the University of
Iowa
Davidson treated mice with
Huntington's
38. Other uses of RNAi
Testing Hypotheses of Gene Function
Target Validation
Pathway Analysis
Studying cell division
Gene Redundancy
Functional Screening
39. Gene Redundancy
In many cases, eliminating the expression of a single gene in
higher eukaryotes can be tolerated even if that gene product
functions in a critical pathway. This is because many critical cell
functions are accomplished by more than one gene product.
When one gene product is eliminated, the redundant gene
product compensates to allow the cell or animal to survive.
Identifying redundant genes could be achieved by co-transfecting
siRNAs and assaying for a given phenotype.
Evaluating each of the candidate genes alone to ensure that they
only cause the cell cycle defect when reduced in combination
with the target gene would help pinpoint the most likely redundant
gene
40. RNA interference characteristics
dsRNA needs to be directed against an exon, not an
intron in order to be effective
Homology of the dsRNA and the target gene/mRNA is
required
Targeted mRNA is lost (degraded) after RNAi
The effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
ssRNA does not work as well as dsRNA
41. Advantage of RNAi
Downregulation of gene expression simplifies "knockout"
analysis.
Easier than use of antisense oligonucleotides. siRNA more
effective and sensitive at lower concentration.
Cost effective
High Specifity
middle region 9-14 are most sensitive
With siRNA, the researcher can simultaneously perform
experiments in any cell type of interest
Can be labelled
Ease of transfection by use of vector
42. Importance of RNAi
Powerful for analyzing unknown genes in sequenced genomes.
efforts are being undertaken to target every human gene via
siRNAs
Faster identification of gene function
Gene therapy: down-regulation of certain genes/ mutated alleles
Cancer treatments
knock-out of genes required for cell proliferation
knock-out of genes encoding key structural proteins
Agriculture
siRNA (small interfering RNA)http://en.wikipedia.org/wiki/Small_interfering_RNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a class
of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes.
They are naturally produced as part of the RNA interference (RNAi) pathway by the enzyme Dicer.
They can also be exogenously (artificially) introduced by investigators to bring about th
knockdown of a particular gene.
siRNA's have a well defined structure.
Briefly, this is a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt overhangs
on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group.
Transfection of an exogenous siRNA is problematic, since it is only transient, and the
dsRNA structure cannot easily be permanently maintained.
One way of overcoming these problems is to modify the siRNA in such a way as to allow it
to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction
of a loop between the two strands, thus producing a single transcript, which can be processed
into a functional siRNA.
This transcription cassette usually uses an RNA polymerase III promoter, which direct the
transcription of small nuclear RNA's, such as U6 or H1. It is assumed (although not known
for certain) that the resulting short hairpin RNA (shRNA) transcript is processed by Dicer.
Introduction of too much siRNA can result in non-specific events due to activation of the interferon
pathway. Most papers suggest that this is probably due to activation of the dsRNA sensor PKR,
although retinoic acid inducible Gene I (RIG-I may also be involved
One method of reducing the non-specific effects is by turning the shRNA into a micro RNA.
Micro RNA's are naturally occurring, and, as such, tolerated better by the cell.
By engineering an siRNA sequence into an miRNA structure, non-specific effects can
potentially be eliminated.
miRNA (micro-RNA)http://en.wikipedia.org/wiki/MiRNA
A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotide long.
It is thought to regulate the expression of other genes.
miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence
includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is
transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base
pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA).
In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA.
The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5,
a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release
the mature miRNA.
In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes
place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.