ווקטורים ויראליים בכליאה ביולוגית ד"ר דליה זגר ראש חידת הבטיחות ממונת בטיחות ביולוגית
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1 ווקטורים ויראליים בכליאה ביולוגית ד"ר דליה זגר ראש חידת הבטיחות ממונת בטיחות ביולוגית
2 Retroviruses הגנום שלהם רנ"א חד גדילי לאחר ההדבקה מתועתק הרנ"א הנגיפי לדנ"א ע"י RT משלבים את הגנום שלהם בדנ"א של התא המאחסן (הכרחי למחזור חייהם)
3 Lentivirus Subfamily of Retroviruses (HIV, FIV, SIV) Lenti - slow (Latin) - long incubation period Most efficient method for gene delivery Exchange env T cells env env of vesicular stomatitis virus (VSVG) wide range of target cells
4 VSVG
5 Practical applications Block the expression of a specific gene (RNAi technology) Introduce a new gene As of today >1000 clinical trials conducted using viral vectors A very useful tool in Molecular Biology
6 Why Lentivirus? What have made them so attractive to use? Insert large genes Target both dividing and non dividing cells Enhanced target range with VSVG Viral genes are deleted and are packaged in a non replicating vectors
7 Produced virus particles are replication deficient RRE Gag-Pol pa Rev pa VSVG pa
8 Produced virus particles are replication deficient RRE Gag-Pol Rev VSVG pa pa pa Producer Infected
9 The attractive features of Lentivirus raises Biosafety issues The risk group are only Laboratory workers handling Lentivirus
10 When??? Producer Infected
11 We do not ignore biohazard we control it Risk Assessment
12 Risk Assessment Lentivirus penetration may occur While handling Through the skin via puncture or absorption (scratches, cuts, dermatitis, or other lesions) Through mucous membrane eyes, nose, and mouth
13 Half life Titer (TU/ml) - 80 o C Freeze- Thaw < Transfection 4 o C 8 days Commercially 22 o C 1-2 days In Vivo 37 o C 56 o C 10 hrs. 10 min ; 10 5 HIV Patients HIV Infective >10 2 dose 70% ETOH 1:10 diluted Sodium Hypochlorite (final con 0.6%)
14 BSL1 agents not known to cause disease (E. coli, Bacillus subtilis) Biosafety Levels BSL2 agents associated with human disease (Human cell lines, Hepatitis B virus, bood born pathogens) BSL3 agents which may cause serious disease (M. tuberculosis St. Louis encephalitis virus) BSL4 agents of life threatening nature (Ebola Zaire,Sin Nombre virus Rift Valley Fever)
15 Lentiviral Vectors BL2+ BL2
16 BL2+ When??? Producer Infected
17 HEPA FILTER (high efficiency particulate air)
18 Sign posted, door preferably closed, # people in the room Pipettes and tips containing viral particles decontamination in the hood Centrifuge in the room (do not leave unattended) No use of Sharps or Glass Tray, Gloves Instruments - clean w 70% ETOH Fluorescent microscope BL2+ Double Gloves, Disposable Lab coat, Sleeves only during virus manipulation, only in the TC room BL2+ Ultra-centrifugation Destabilize at ph<6 or ph>8 After one week Show and Tell
19 Adenoviruses Group: Group I (dsdna) Family: Adenoviridae DNA Viruses Medium-sized ( nm) Nonenveloped viruses מתמירים תאים של מכרסמים
20 Adenoviruses Approximately 50 different serotypes most common Type 5 Adenoviruses can cause a spectrum of illnesses; common cold, pink eye, diarrhea, in rare cases - Hepatitis Rarely integrate into the chromosome Infects many cell types and resting cells Generate immune response (כליאה ביולוגית ( work Relatively safe at lab
21 כליאה ביולוגית -אדנווירוס Adenoviral vectors derived from type 5 adenovirus First generation vectors Two early genes deleted : E1 or E1 and E3 regions E1 deletion renders the virus incapable of reproducing itself E3 deletion renders it more susceptible to immune defense system This is also the region for transgene insertion Second generation vectors Deletion of : E1, E3,E4 Third generation vectors Gutless vectors all viral genes deleted (only essential cis-acting seq retained)
22 Adeno-Associated virus (AAV) Group: II (ssdna) Family; Parvoviridae וירום קטן 20 - ננומטר, ללא מעטפת נמצא לראשונה במהלך הפקת Ad קיומו כוירוס אינפקטיבי תלוי ב helper virus לכן נקראים Dependovirus הוירוס אינו משתכפל באופן עצמאי - כליאה ביולוגית יכול להדביק סוגי תאים רבים heparan sulphate proteoglycans הדבקת אדם אינה סימפטומטית
23 Adeno-Associated virus (AAV) 4.7kb ITR Rep Cap ITR ITR ; 145 nucleotide, inverted terminal repeats Rep (78, 68, 52, 40) ; Non structural- viral replication and packaging Responsible for site specific integration (of wt) Cap (VP1-3) ; Structural - capsid proteins Production of the recombinant vector requires that ; rep & cap are provided in trans, along with helper virus gene products (E1a, E1b, E2a, E4 & VA RNA from Ad. genome) החסרון גודל האינסרט
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