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Genome Biology Unit (GBU)
Institute of Biotechnology, Viikki
Tea Vallenius, Kaisa Laajanen, Niina Roine
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Content
• General view• Description of our libraries• Basics of Gateway cloning
• How to order• Clones and cloning service• Future plans (e.g. kinome sets, custom projects)
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
http://www.biocenter.helsinki.fi/bi/gbu/
GBU services 2011 - 2013
- 28 research groups of which 12 represent new groups in 2013
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Value for scientists in Finland!
- reduced costs
- rapid delivery
- easy access
- screening possibilities
MGC Collection. cDNA library
Human 20 945
Mouse 8 290
ORFeome Collaboration. Full ORFs
Human 16 934
Mouse 1 758
cDNA/ORF sets available through Genome Biology Unit
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
MGC collection = Mammalian Gene collection
- National Institutes of Health initiative started 2001. The aim was to generate full-length protein coding cDNAs for human and mouse genes
- sequence validated
- MGC clones were intially obtained by random EST-screening of cDNA libraries
ORF5’ UTR 3’ UTR
- From the practical point of view:
*clones we have are in various vectors
-> not necessarily suitable for your studies immediately
-> more time-consuming & expensive to transfer to other vectors
*full length cDNA clones
-> may contain 5’/3’ UTRs, which may have a regulatory role
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
http://mgc.nci.nih.gov/
The ORFeome collection
-ORFeome Collaboration, which aim is to provide sequence validated, ORF clones in Gateway entry vectors allowing easy transfer of the ORF sequences into various expression vectors
-many available either with or without stop codons
-From the practical point of view:
*prior to expression studies need to be transferred into destination vectors
*easy to transfer to any destination vector using Gateway cloning
*contain only the protein coding sequences
*GBU’s library has been intially validated
ORF
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
I. BP reaction = “ insert your gene of interest into the Gateway Technology”
2. negative selection3. specific enzyme
ORFORF
1.site-specific recombination
kanakana
Basics of the Gateway technology: Generation of an entry clone
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
I. BP reaction = “ insert your gene of interest into the Gateway Technology”
2. negative selection3. specific enzyme
ORFORF
1.site-specific recombination
kanakana
Basics of the Gateway technology: Generation of an entry clone
ORF
entryclone
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
2. LR reaction = “ transfer your gene of interest into destination vectors”
3. specific enzyme
Basics of the Gateway technology: Generation of an expression clone
2. negative selection
ORFORF
1.site-specific recombination
amp amp4. antibiotic selection
Cloning service provided by GBU!
= OFR library
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
2. LR reaction = “ transfer your gene of interest into destination vectors”
3. specific enzyme
Basics of the Gateway technology: Generation of an expression clone
2. negative selection
ORFORF
1.site-specific recombination
ampamp4. antibiotic selection
Several advantages:
- rapid to perform
- efficient : > 90% success rate
- universal : no need to consider cloning sites - reversible : entry -> destination -> entry
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Content
• General view• Description of our libraries• Basics of Gateway cloning
• How to order• Clones and cloning service• Future plans (e.g. kinome sets, custom projects)
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
http://www.biocenter.helsinki.fi/bi/gbu/
• Available clones on excel sheets • Ordering form • Sequence your clones easily
– Collaboration with DNA sequencing and Genomics laboratory in Viikki and Biomedicum Sequencing Unit (BSU)
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
How to order: available ORF/cDNA sets
How to order: available ORF/cDNA sets
• Available clones on excel sheets
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
How to order: ordering form
• Available clones on excel sheets • Ordering form • Sequence your clones easily
– Collaboration with DNA sequencing and Genomics laboratory in Viikki and Biomedicum Sequencing Unit (BSU)
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
How to order: we recommend to verify you clones
• Available clones on excel sheets • Ordering form • Sequence your clones easily
– Collaboration with DNA sequencing and Genomics laboratory in Viikki and Biomedicum Sequencing Unit (BSU)
– Primers available for verification of ORF clones
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
PetriAuvinen
LarsPaulin
TapioTainola
ORF cloning service
• We clone your ORF clones of interest to multiple Gateway destination vectors • Cloning format: Single clones or 96-well format• Remember: FuGU can provide viral particle production service from your lenti-viral
constructs
Most commonly used Gateway destination vectors:
N- terminal GST
N- terminal 6xHis
C- terminal V5 + 6xHis
C- terminal GFP
Lenti vector with C- term V5 Stop
N-terminal emGFP + C- term V5,C-term emGFP
N- and C- term. mCherry (a kind gift from Maria Vartiainen)
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Mammalian Expression
Bacterial Expression
Insect Expression
Entry Clone
96-well cloning and transfections trials
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Pictures taken with Cell Insight in Light Microscopy Unit (LMU)
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
GBU/FIMM/FuGU joined forces: - GBU is currently generating human Kinome ORFs (FIMM & FuGU have kinome knockdown reagents)
Custom projects- Custom-tailored Gateway clonings- Primer script to design primers with Gateway tails- If you have ideas, we are happy to discuss
Future Plans
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
New service available
- 3DHISTECH Pannoramic digital slide scanner
New service available
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014
Genome Biology Unit
Contact information:
Phone: 09-19159429Email: [email protected] pages: www.biocenter.helsinki.fi/bi/gbu
Personnel:Niina Roine M ScKaisa Laajanen M ScTea Vallenius MD, PhD
Genome Biology UnitInstitute of Biotechnology
Genome scale reagents and services for functional studies
22.01.2014