genome biology unit (gbu) institute of biotechnology, viikki tea vallenius, kaisa laajanen, niina...

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)



22.01.2014



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



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



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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



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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



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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



22.01.2014

I. BP reaction = “ insert your gene of interest into the Gateway Technology”

2. negative selection3. specific enzyme

ORFORF


kanakana

Basics of the Gateway technology: Generation of an entry clone

ORF

entryclone



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


amp amp4. antibiotic selection

Cloning service provided by GBU!

= OFR library



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


ampamp4. antibiotic selection

Several advantages:

- rapid to perform

- efficient : > 90% success rate

- universal : no need to consider cloning sites - reversible : entry -> destination -> entry



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)



22.01.2014



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)



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How to order: available ORF/cDNA sets

How to order: available ORF/cDNA sets

• Available clones on excel sheets



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How to order: ordering form





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How to order: we recommend to verify you clones



– Primers available for verification of ORF clones



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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)



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Mammalian Expression

Bacterial Expression

Insect Expression

Entry Clone

96-well cloning and transfections trials



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Pictures taken with Cell Insight in Light Microscopy Unit (LMU)



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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



22.01.2014

New service available

- 3DHISTECH Pannoramic digital slide scanner

New service available



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



22.01.2014

mailto:[email protected]

mailto:[email protected]

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