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Gmo.hr

Federal Ministry of Health and
Women - Division IV/B/12
Radetzkystraße 2
A - 1030 Wien
November 2003

NOTIFICATION
of work with GMO
large scale
(tick where applicable)
Classification of the intended work (tick where applicable)

Biosafety level 1
Biosafety level 2
Classification of GMO (tick where applicable)

bacteria
other eucaryotic cells
agricultural crops
other plants
other microorganisms
farm animals
human cell tissue
other animals

Work with GMM:

culture volume:
500 ml - 1 l
more than 600 liter ( l)
(declare volume)

minutes of the BSC attached:

Informations on the carrier, BSC, BSO, project manager and premises:
carrier:
Company A
address:
Street B 1 A-1020 Vienna
phone: 123456
fax:

e-mail: office@companyA.at
biological safety officer (BSO):
phone: 123456
fax:

e-mail: Dr.B@companyA,at
qualification and education:

Ph.D. in Genetics, University of Vienna
3 years postdoc, University of Graz
5 years project manager Comp. D
for more details see the attached curriculum vitae
1.2.1. deputy of the BSO:
name:

phone: 123456
fax:

e-mail: Dr.C@companyA.at
qualification and education:

Ph. D. in Biochemistry and Biotechnology; University
postdoc,
University
for more details see the attached curriculum vitae
members of the biological safety committee (BSC):
1.3.1. number:
overall: 4
internal: 3
external: 1
1.3.2. internal members:
name:

qualification and education:
see 1.2

qualification and education:
see 1.2.1

qualification and education:

Ph. D. in Microbiology, University of Texas
10 years Biosafety Officer of Comp. G
for more details see the attached curriculum vitae
qualification and education:

qualification and education:

1.3.3. external members:
name:

Prof. Dr. E
address:
Inst. of Microbiology, University of Vienna, A-1030
phone: 112233
fax:

e-mail: Dr.E@mibi.univie.ac.at
qualification, education and present occupation:
Ph.D.

Biotechnology,
University
since 10 years , a. o. Prof. University of Vienna
for more details see the attached curriculum vitae
address:
qualification, education and present occupation:

project manager (not applicable for work with GMO in biosafety level 1):
phone: 123456
fax:

e-mail: Dr.F@companyA.at
qualification and education:

Ph D. in Genetics, University of Vienna
postdoc,
University
for more details see the attached curriculum vitae
information on the premises:
1.5.1. adress of the premises:
Street B 1, A-1020 Vienna

1.5.3. description of the parts of the premises, relevant for the work with GMO and for
safety (including attached construction plans, as well as description of the equipment
according to Annex II of the ordinance on work with GMO in the contained use; BGBl.
II Nr. 431/2002):

Room Nr. 1001: PCR-Machine, centrifuges, EB-bath
Room Nr. 1002: gel-documentation, photometer, speed vac
Room Nr. 1003: laboratory, laminar-flow class 2, -20°C and 4°C refrigerator
Room Nr. 1004: sterilisator, autoclave, dishwasher
Room Nr. 1005: -80°C refrigerator, ultracentrifugation
Room Nr. 1006: microscopy
Room Nr. 1007: incubator room 37°C
Room Nr. 1008: incubator room 28°C
The walls, floors, and working benches are impervious to water, and resistant to
acids, alkalis, solvents and disinfectants. In the laboratory there is a hand wash
basin with dispensers containing soap and hand disinfectant, respectively, as well
as a dipenser for paper towels.
for more details see the attached maps
summarized description of the work:
title of the work:

Cloning of genomic DNA from HIV-1 in E.coli K12, Cos7-, H9- and Jurkat- cell lines

description of work/fermentation process including expected results with
relevance according to biosafety issues (detailed and chronological description of
the intended work):


1. Cloning of HIV-1-gag, env,tat and nef into H9 and Jurkat cell lines:

HIV- genes will be amplified by PCR, modified by adding 5’ an EcoRI
and 3’ a HindIII restriction site and cloned into pAB12 (which was derived from
pBR322 by inserting an modified MCS and the RSV LTR promotor from
pRSVneo).

H9 and Jurkat cells will then be transformed with the resulting pAB12env,
pAB12gag, pAB12tat and pAB12nef plasmids

2. Cloning of HIV-1 total genome into E. coli K12:
Total genomic DNA of HIV-1 will be isolated and cloned into pUC19 (pBR322
derivate). Then E. coli K12 will be transformed with the resulting plasmid
pUC19HIV

3. Cloning of HIV-1- total genome into Cos7-cells:
Total genomic DNA of HIV-1 will be isolated and cloned into pBR322. Then Cos7
cells will be transformed with the resulting plasmid pBR322HIV.
description of the recipient organism (e. g. genotype, biosafety level, source,
citations, catalogue number ans other risk assessment relevant properties) :

E. coli K-12 ATCC10798, biosafety level 1
E. coli K-12 is apathogen und a laboratory strain. Because of its auxotrophy this
strain is not able to survive in the environment.
COS 7 ATCC CRL-1651, biosafety level 2
H9 ATCC HTB-176, biosafety level 1
Jurkat DSMZ ACC282, biosafety level 1
description of the donor organism (e. g. genotype, biosafety level, source, citations,
calogue numbers and other risk assessment relevant properties):


HIV-1, biosafety level 3

description and designation of used vectors including risk assessment relevant
properties (e. g. antibiotic markers, promoters, origins of replication, repressors,
biosafety level, source, citations, catalouge numbers and other risk assessment
relevant properties):

pUC19: this vector is an derivate of the biosafety vector pBR322 and contains
an ampicilin resistance, the lac promotor , the lac Z’ gene, and MCS and the
pMB1 ori. For more details see the attached vector map
pAB12: this vector was obtained from pBR322 by inserting the RSV LTR
promoter

from pRSVneo into pBR322. It contains an ampiciline and a tetracycline
resistance

the pMB1 ori and a modified MCS. As this vector is a derivate of the biosafety
vector pBR322 it could be also classified into biosafety level 1.
For more information see the attached vector maps.
pAB12gag, pAB12env, pAB12tat and pAB12nef are derivates from pAB12
containing the respective genes from HIV-1 and should be therefore biosafety
pUC19HIV contains the total HIV-1 DNA.
description and designation of the material and gene products used for genetic
manipulation (e. g. sequence data, accession numbers, biosafety level, source,
citations and other risk assessment relevant properties):

HIV-1 genes gag, env, tat and nef as well as total HIV-DNA. Although HIV-1 is
classified into biosafety level 3, the use of an biological safety system (E. coli K12)
and derivates of the biosafety vector pBR322 allows to classify this work into
biosafety level 2
Argumentation of the risk assessment with reference to human beings, animals,
plants and environment (e. g. use of an approved biological safety system, no
advantage for the survival of the GMO in the envirnonment compared to the
recipient, no toxicity of the gene product etc.):

1. Cloning of HIV-1-gag, env, tat and nef into H9 and Jurkat:
Althoug the donor organism is classified into biosafety level 3, the use of
defined and characterised genes together with the use of an approved biosafety
system (pBR322 derivates, recipient organisms of biosafety level 1) results in a
downgrading of the biosafety level to biosafety level 2
2. Cloning of total HIV-1 DNA into E. coli K12:
Although total DNA of an biosafety level 3 organism will be cloned, the use of
E. coli K12 (biosafety level 1) combined with the use of an pBR322 Derivate
resembles an approved biological safety system. Therefore this part of the work
will be classified into biosafety level 2
3. Cloning of total HIV-1 DNA into COS 7 cells:
Since the used plasmid pBR322 does not posess an eukaryotic promoter and
of the immobilization of this plasmid, but based on the fact, that the donor
organism

is classified level 3 and the recipient is classified on biosafety level 2, this part
of the work will be classified into biosafety level 2
Classification of the GMO into a biosafety level group and description of the
intended safety precautions (e. g. compliance of international GLP-/GMP-
standardsm instruction of the personal, restricted access to laboratories where
GMO are handled etc.):

The GMOs are classified into biosafety level 2.
All work where aerosols can be produced will be done in an class 2 biosafety
cabinet. All work will be done in compliance of international GLP standards.
The personal working with GMO will be instructed properly. A hygiene plan is
provided. Also there is restricted access to the laboratory
2.9. description of the measures for the inactivation of GMO and the waste managment:
All material, glassware and cultures will be inactivated by autoclaving for 30 min. at
121° C. The benches will be routinely cleaned with appropriate disinfectants (e. g. Et-
OH, Na-Hypochloride).
All inactivated waste will be collected in sealed barrels and incinerated

declaration of the containment level to verify the biosafety level according to Part B Z 3
of the ordinance of work with GMO in the contained use, BGBl. II Nr. 431/2002 (tick
where applicable):

Einschließungsstufe 1
Einschließungsstufe 2
2.11. classification of the intended work into biosafety level (tick where applicable):

biosafety level 1
biosafety level 2
3. safety precautions:

3.1. information on the rules for accident precaution (only for work in biosafety level

All work will be done in biosafety level 2 equipped laboratories. Process steps where
hazardous quantities of aerosols are formed will be done in class two biosafety cabinets.
The personal will be instructed properly. There will be restricted access to the
laboratories

4. internal
enabling:
Assessment of the biological safety committee (BSC):
the classification of the GMO into the biosafety level mentioned at point 2.11 was
affirmed (tick where applicable):

minutes of the BSC are attached (tick where applicable)
following information should be handled confidential according to § 105 and §
106 of the Austrian Gene Tecnology act and should therefore not become public:

siganture of the CARRIER:

date: 05.05.2004
signature:

Source: http://www.gmo.hr/cro/content/download/297/1707/file/Notification%20of%20work%20with%20GMO%20-%20level%202.pdf

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