What's New at the Vector Core

What's Old is New Again: Subcloning in the Vector Core

BY TOM LANIGAN, Ph.D., Director, Vector Core

MEET THE VECTOR CORE

Roland Holgarth, Ph.D., Subcloning, Repository, BacMam

Huira Kopera, Ph.D.

CRISPR Cell Lines

Tom Lanigan, Ph.D.

Director

Tonya Kopas, M.S.

Lentivirus

Bob Agate, Ph.D.

AAV

OVERVIEW OF SERVICES

• New Viral Products
  • Adeno-Associated Virus (AAV)
  • Rabies-Tracers
  • Bac-mam
• Repository Expression
  • Plasmids Viral-Based
  • Plasmid (Lenti, Retro, AAV) CRISPR Plasmids
• Plasmid Construction (Subcloning Services)
• Custom CRISPR cell lines

Adeno-Associated Virus (AAV)

• In Vivo Transduction
• Persistent expression in non-dividing cells
• No Replication Competence
• Low immunological response
• ~4.7kb limit
• Safe - Generally BSL1 containment

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Adeno-Associated Virus (AAV)

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Figure from Addgene

Adeno-Associated Virus (AAV) Products

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Custom Virus

• Serotypes 1, 2, 5, 6, 6.2, 7, 8, 9, PHP.eB, PHP.S, retro
• Three Scales – 50 ml, 300 ml and 1ml at ~1013 vg/ml

Stock Virus

• 50 ml at ~1013 vg/mlSampler of AAV1, 2, 5, 8, 9 (10 ml each)
• Various
  • promoters: CMV, CAMKIIa, UBC, EF1a, (CAG, hSYN – in process)
  • reporters: eGFP, mCherry, and CRE
  • In developmental stage to build repository of AAVs that are of interest to U-M Investigators. 
    • e.g. TBG-driven reporters for expression in the liver

Rabies Products

• Maps neuronal circuits in brain
  
• Spread by retrograde transport between neurons
• Targets specific neurons In Vivo Transduction

Wall et al. 2010 PNAS

Baculovirus/Bac-mam System

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Advantages 

• Large Packaging Limit
• Transient, High Expression
• Co-transduction
• No Replication Competence
• Low immunological response
• Safe- BSL1 containment

Photo: Mansouri et al. 2016 Nat. Com.

Disadvantages

• Inactivated by Blood Complement

Baculovirus/Bac-mam System

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Advantages 

• 1-2 week turnaround time
• Half the cost of outsourcing
• Materials stay at U-M
• Personalized service
• All Constructs can go into the repository

Vector Core Repository + Subcloning

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

How can the Vector Core Help Your Research?

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

You

Vector Core

• Tell us your research background
• Tell us your project needs and reagents

• Vector Core services/customization
• Potential pitfalls/caveats
• Timeline and Cost

How can the Vector Core Help Your Research?

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

Tonya Kopas, M.S.

Lentivirus

CRISPR-edited Custom Cell Lines

Known cell lines: 

• that can grow from a single cell 
• has intact NHEJ and HDR 
• decent transfection/electroporation efficiency  
• known karyotype 
• known allele number 
• mycoplasma-free

You

Vector Core

• Custom cell line with specific mutation, tag, KO

• CRISPR-edited cell line with mutation under endogenous conditions

Summary

Currently Available

Coming Soon

• AAV
  • currently available, building stock repository
  • Rabies (GFP, mCherry) and self-inactivating rabies available
• Subcloning services

• Plasmid Repository
• CRISPR-edited custom cell lines
• TBD: Baculovirus/Bac-mam

Acknowledgements

Thank you to:

• BRCF Administration
  • Cassie Wong,
  • Anitha Chennat
• Vector Core
  • Tom Lanigan, Tonya Kopas, Roland Holgarth, Bob Agate

• Collaborators
  
  • Martin Myers, Dawen Cai
• Special Thanks to:
  • Bishr Omary