This thesis aimed to characterize the functional regions of the varicella-zoster virus encapsidation proteins using two strategies. The first strategy was to generate stable cell lines expressing each encapsidation protein to use in complementation assays with VZV mutants. Some progress was made generating cell lines expressing ORF30, ORF25 and ORF54. The second strategy was to generate an ORF54 transposon library to map essential and non-essential regions of the portal protein. Significant progress was made preparing the ORF54 vector for mutagenesis. Overall, the work laid the foundation for future functional studies of the encapsidation proteins through cell line complementation and transposon mapping.
Development of Dot-blot Hybridization Based on 522 bp Repetitive Sequence (R5...Tenri Ashari Wanahari
Toxoplasmosis, arising from infection by Toxoplasma gondii, is one of the most common parasitic diseases in humans and other warm-blooded animals. In humans, infections are usually asymptomatic but severe disease can occur in immunocompromized individuals and newborns. Due to the importance of the disease and in order to take suitable measures, an early diagnosis of the disease is essential, particularly for pregnant women and in industry of domestic animals. The genome of T. gondii contains repeat sequences B1 and R522 which constitute ideal targets for genome-based detection methods. The 522 base pairs repeat sequences R522 are the most promising due to the high copy number, evaluated to be 200 to 300 units within the genome. We developed a simple dot-blot hybridization based on R522 sequences. The method is simple and does not require sophisticated devices. The test of the method, using cloned R522 as target, showed that the parasite detection method was sensitive and proved to be promising for use in routine health controls as well as for the survey of Toxoplasma infections.
Key words: DIG-probe, dot blot hybridization, repeat sequences, R522, Toxoplasma gondii.
Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase
Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA
Use of reporter genes in the process of selection of the transformants from the non transformants, and the current use of these reporter genes as the Desired genes.
Development of Dot-blot Hybridization Based on 522 bp Repetitive Sequence (R5...Tenri Ashari Wanahari
Toxoplasmosis, arising from infection by Toxoplasma gondii, is one of the most common parasitic diseases in humans and other warm-blooded animals. In humans, infections are usually asymptomatic but severe disease can occur in immunocompromized individuals and newborns. Due to the importance of the disease and in order to take suitable measures, an early diagnosis of the disease is essential, particularly for pregnant women and in industry of domestic animals. The genome of T. gondii contains repeat sequences B1 and R522 which constitute ideal targets for genome-based detection methods. The 522 base pairs repeat sequences R522 are the most promising due to the high copy number, evaluated to be 200 to 300 units within the genome. We developed a simple dot-blot hybridization based on R522 sequences. The method is simple and does not require sophisticated devices. The test of the method, using cloned R522 as target, showed that the parasite detection method was sensitive and proved to be promising for use in routine health controls as well as for the survey of Toxoplasma infections.
Key words: DIG-probe, dot blot hybridization, repeat sequences, R522, Toxoplasma gondii.
Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase
Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA
Use of reporter genes in the process of selection of the transformants from the non transformants, and the current use of these reporter genes as the Desired genes.
this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
Main file> http://www.slideshare.net/rustradeESP/testgene
TestGene develops and manufactures kits for molecular genetics. Products are intended for use in research, practical medicine, and in the fields of molecular biology. The focus area – non-invasive genetic testing in obstetrics and oncology. The company has its own production laboratory with the necessary equipment for manufacturing and quality assurance of kits based on "real time PCR".
Delivering More Efficient Therapeutic Protein Expression Systems Through Cell...Merck Life Sciences
Historically cell line performance has been enhanced through media, feed and process optimization, primarily through trying to meet the basic nutritional requirements of the cells so that they can sustain high growth and productivity throughout the production runs.
However, the omics (genomics, transciptomics and metabolomics) era, sequencing of the CHO genome and enhancements in genome editing technologies over the past several years have enabled scientists to take a more direct route in cell line optimization through the modification of specific genes that have direct implications on cell culture performance, protein quality attributes and upstream and downstream manufacturing processes. These targets include but are not limited to genes that may be involved in cell cycle regulation, cellular metabolism, cellular transcription and translation, the secretory pathway and protein glycosylation or other post-translational modifications.
In this webinar we will discuss specific genetic modifications that have been made to CHO cell lines and how these modifications can lead to more efficient expression systems.
Herpes zoster by dr bashir ahmed dar associate professor medicine sopore kas...Prof Dr Bashir Ahmed Dar
Herpes zoster (or simply zoster), commonly known as shingles and also known as zona, is a viral disease characterized by a painful skin rash with blisters in a limited area on one side of the body, often in a stripe. The initial infection with varicella zoster virus (VZV) causes the acute (short-lived) illness chickenpox which generally occurs in children and young adults.
this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
Main file> http://www.slideshare.net/rustradeESP/testgene
TestGene develops and manufactures kits for molecular genetics. Products are intended for use in research, practical medicine, and in the fields of molecular biology. The focus area – non-invasive genetic testing in obstetrics and oncology. The company has its own production laboratory with the necessary equipment for manufacturing and quality assurance of kits based on "real time PCR".
Delivering More Efficient Therapeutic Protein Expression Systems Through Cell...Merck Life Sciences
Historically cell line performance has been enhanced through media, feed and process optimization, primarily through trying to meet the basic nutritional requirements of the cells so that they can sustain high growth and productivity throughout the production runs.
However, the omics (genomics, transciptomics and metabolomics) era, sequencing of the CHO genome and enhancements in genome editing technologies over the past several years have enabled scientists to take a more direct route in cell line optimization through the modification of specific genes that have direct implications on cell culture performance, protein quality attributes and upstream and downstream manufacturing processes. These targets include but are not limited to genes that may be involved in cell cycle regulation, cellular metabolism, cellular transcription and translation, the secretory pathway and protein glycosylation or other post-translational modifications.
In this webinar we will discuss specific genetic modifications that have been made to CHO cell lines and how these modifications can lead to more efficient expression systems.
Herpes zoster by dr bashir ahmed dar associate professor medicine sopore kas...Prof Dr Bashir Ahmed Dar
Herpes zoster (or simply zoster), commonly known as shingles and also known as zona, is a viral disease characterized by a painful skin rash with blisters in a limited area on one side of the body, often in a stripe. The initial infection with varicella zoster virus (VZV) causes the acute (short-lived) illness chickenpox which generally occurs in children and young adults.
Recombinant viral vectors are genetic engineering tools commonly used for gene transfer purpose with high transfection efficiency and site specific gene insertion.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
*PROJECT ON LUCIFERASE GENE CLONING
1. SUITABLE VECTOR
2. RESTRICTION ENZYMES ALONG WITH THEIR RESTRICTION SITES
3. CLONING PROCESS
4. EXPRESSION
5. PGL-2 CONTROL VECTOR DIAGRAM
6. COMPARISON OF VECTOR IN MORE THAN ONE HOST
7. NEW INNOVATIVE IDEA FOR CLONING
8. CONCLUSION
9. SUMMARY
This lecture is about Virology of HCV presented by Dr. Mahmoud Elzalabany, Internal Medicine Resident, Ahmed Maher Teaching Hospital.
The lecture was presented in the scientific meeting of Internal and Tropical Medicine departments, Ahmed Maher Teaching Hospital titled (Towards Eradication of HCV in Egypt) in celebration of World Hepatitis Day on July 28, 2016.
https://www.facebook.com/AMTH.IM
https://www.facebook.com/events/1072758396145209/
http://www.no4c.com
Molecular markers- RFLP, RAPD, AFLP, SNP etc.Cherry
Molecular markers are identifiable DNA sequences used to locate genes associated with specific traits or genetic conditions.
A molecular marker is a specific gene fragment present at a specific position called ‘locus’ (pleural loci) in the genome of a cell.
In the pool of unknown DNA or in a whole chromosome, these molecular markers help in identification of particular sequence of DNA at particular location.
Similar to Strategies to Characterize Functional Regions of the Varicella-Zoster Virus Encapsidation Proteins (20)
9. PURPOSE OF THESIS
• Two strategies:
• 1) Stable cell lines expressing encapsidation proteins
• 2) Encapsidation protein transposon libraries for use in
functional studies
• Hypothesis: specific regions of encapsidation
proteins will be essential for DNA encapsidation
since it is known that any one deletion of HSV-1
homologs results in accumulation of empty capsids
and un-cleaved DNA in the nucleus
10. STRATEGY 1: CELL LINE
COMPLEMENTATION
• Cell line complementation as a tool to study
encapsidation gene mutants
• HCMV, HSV-1, PRV
• Hypothesis: deletion of each putative DNA
encapsidation gene will yield a phenotype
consistent with a defect in cleavage and/or
packaging
• Essential to this process is the isolation of
stable, DNA encapsidation protein expressing cell
lines
11. CELL LINE COMPLEMENTATION
Deleted gene
VZV Mutant
Cell expressing deleted gene
• VZV genome without one encapsidation gene
• VZV requires each encapsidation gene for replication
12. MEWO CELL LINE
• Previously, each encapsidation ORF was transiently
expressed in MeWos (Visalli, 2007)
• Stable cell line production
• Geneticin sensitivity assay for LC75
• Transfection of ORF30 & ORF54 vectors
14. MEWO CELL LINE
• Transfection of ORF30 & ORF54
• Indirect immunofluorescence (IF)
• 6, 15, & 27 d
• Western Blot at 1 month
• confirmed IF results
15. FLP-IN CV-1 CELL LINE
• Flp-In two plasmid system
• Designed for rapid generation of stable cell lines
• Utilizes an FRT site & has reporter genes
• Successful integration of ORF means cells are:
• zeocin sensitive
• hygromycin resistant
• lacks B-gal activity
• expresses ORF
http://products.invitrogen.com/ivgn/product/K601001
16. FLP-IN CV-1 CELL LINE
• Complementation is
dependent on VZV
replication
• Can VZV grow in
CV-1?
• Plaque assay from 7 d
infection
• IF from 10 d infection
17. FLP-IN CV-1 CELL LINE
• Transient expression of ORF25, ORF54, & ORF30
• IF at 3 to 4 d post transfection
18. ORF30 TRANSFECTION
• 3 variables:
DNA, Lipofectamin
e, Hygromycin
• Only 6 colonies
survived
19. ORF 30: B-GAL ASSAY
• 6 ORF30 colonies
were assayed for
B-gal activity at 1
and 3 months
20. ORF30: PCR
• Presence of ORF30 gene in ORF30 cell lines were
tested by PCR
• 770 bp or 0.7 kbp
28. DISCUSSION
• VZV ORF expressing FlpIn cell lines isolated?
• Hygromycin resistant
• Lacked B-gal activity
• PCR
• Indirect immunofluorescence
• Western blot
• Immunoprecipitation followed by Western blot
• Complementation
29. DISCUSSION
• Stable cell lines must have ORF incorporated in
genome
• A rare event
• Promotors of ORF versus reporter genes
• Cellular toxicity
• Inducible promotor
• Improper ORF orientation
• Using complementation as selection
30. DISCUSSION
• Isolate encapsidation gene expressing cell lines
• Grow VZV mutants for functional studies
• Experimentally prove that the ORF is essential
• Deficient in DNA cleavage versus DNA translocation
31. STRATEGY 2: ORF54 TRANSPOSON
LIBRARY
• Little is known about the role of specific functional
regions within the pORF 54 portal
• ORF54 transposon library as a tool to study portal
function (pORF54)
• Portal isolation in HCMV, HSV-1, VZV
• Hypothesis: specific regions of pORF54 will be essential
for interaction with the viral capsid, terminase complex,
and viral DNA.
• Essential to this is a ORF54 vector prepared for
mutagenesis for the use in functional studies
32. CONSTRUCTION OF
ORF54 VECTOR
• 3165 bp
23130
9416
6557
4361
2322
2027
564
http://tools.invitrogen.com/content/sfs/vectors/pcr4blunttopo_map.pdf
33. CONSTRUCTION OF
ORF54 VECTOR
• Transformation of ORF54 vector into E. coli
• EcoR1 digestion
Not to scale
34. MANIPULATION OF
ORF54 VECTOR
• Removal of Not1 site
• Digested with Not1 Restriction
Enzyme
• Termination End Reaction
• Repair sticky ends to blunt
ends
• Resulted in the deletion of
Not1
• Ligation
• Transformation of ORF54
minus Not1
Transformation ORF54 without Not 1 site
35. VERIFICATION OF
ORF54 VECTOR
• Selection for Not1 Deletion
Undigested versus Not1 digested ORF54 vector
39. MUTAGENESIS
Epicentre EZ-Tn5 In-Frame
Linker Insertion Kit was used
to randomly insert mutations
on our ORF54 vector via
transposons
http://www.epibio.com/item.asp?id=289
41. MUTAGENESIS
Plasmid DNA was isolated from individual E.coli transformants
Digestion with Not1 to remove transposons
Removal of Transposon with Not1 digsetion Gel of Not1 digested mutated ORF54
http://www.epibio.com/item.asp?id=289
42. DISCUSSION
• Prepared the foundation for ORF54 transposon
library
• Isolated our ORF54 fragment
• Prepared the ORF54 vector by deleting Not1 and Kan
• Vector is ready for mutagenesis to make transposon library
for future functional analysis
• pORF54 regions can then be characterized by
complementing ORF54 VZV BAC
• ORF54 versus mutated ORF54
• Map essential and nonessential regions
43. THESIS CONCLUSION
• Characterizing functional regions will reveal the
mechanisms of DNA cleavage and packaging
• Both strategies would prove encapsidation proteins are
essential for VZV replication
• Hopefully these studies will get us closer to
understanding encapsidation
44. THANK YOU
• Dr. Visalli
• Vi Tran
• Rick Covington
• Dr. Hua Zhu (Rutgers)
• Funding from NIH R15 AI062713-02
Editor's Notes
1) Dissociation of tegument (pink bubbles), 3)Concatameric DNA, 5)Virion picks up glycoproteins from Golgi body before exiting
Spp1
Complementation of an encapsidation mutant virus depends on whether VZV replication can be supported by CV-1 cells
9 hour transfection. 3 days cells were diluted and 17 days post transfection, counted. At 22 days, transferred colonies. 200 ug/ml was decreased to 100.
The lack of protein expression was perplexing. it was difficultto find GFP expressing CV-1 cells from the ORF30BAC virus. There were only single cells expressing GFP and no obvious clusters of GFP cells, which would be an indication of plaque formation and spread, were observed.
25: 22 kda54: 91 kda30: 92.1
There were single cells expressing GFP (Figure 14D) and some larger clusters of GFP expressing cells (potential plaque formation; Figure 14C) suggesting that the mutant ORF54 genome was complemented.