Cloning Club: Shuttle Vectors Bacteria E.coli, Bacillus subtilis, Lactococcus, Pseudomonas Yeast Saccharomyces cerevisiae, Pichia pastoris Insect cells Drosophila (Schneider S2), Sf9, High5 Chemical Protein Synthesis Mammalian cells HEK293, CHO Cell-free Expression Merrifield 163, first sythetic protein 1969

Shuttle Vectors A shuttle vector is a vector that can propagate in two different host species, typically E. coli and a eukaryotic host species. mammalian cells insect cells E. coli yeast

ori = host cell-specific site where the DNA replication is initiated E. coli origin of replication ColE1 ori: 15-20 copies/cell (e.g. pBR322, „low copy plasmid“) pUC ori (= mutated ColE1): 500-700 copies/cell („high copy plasmid“) f ori (BACs): 1 copy/cell SV40 origin of replication

SV40 ori From SV40 polyoma virus SV40 ori-containing plasmids replicate in cells that are a) infected with SV40 polyoma virus b) express the large T antigen (HEK) 293T cells (these cells are stably transfected with a large T antigen expressing construct, which was selected with G418/Geneticin) “Episomal” replication: DNA doesn’t become diluted with cell division (as quickly as non- replicating plasmids)

EBNA ori From Epstein-Barr virus oriP 293EBNA cells Requires EBNA-1 gene product Does work only in human cells

E. coli-yeast shuttle vectors For propagation in E. coli and Saccharomyces cerevisiae Used for reliable assembly of large DNA constructs (yeast recombinational cloning, YRC): http://www.labtimes.org/labtimes/method/methods/2010_03.lasso

E. coli-mammalian shuttle vectors Most of them a based on DNA viruses with a circular genome, to which a bacterial ori and selection marker have been added Adeno-associated virus (AAV): ~4.8 kb Adenovirus (AV): ~36kb Baculovirus (AV): ~130 kb insect cells

“Shuttle” vector for AAV production ITRs functionally replace the ori

“Shuttle” vector for AAV production capsid proteins determine what cells are infectable (“tropism”) Büning et al. Recent developments in adeno-associated virus vector technology. Journal of Gene Medicine. 10, 2008: 717–33.

True “shuttle” vector? Adenovirus (AV) Adeno-associated virus (AAV) type pathogenic non-pathogenic non-enveloped replicating replication-defective genome 26-48 kb dsDNA 4.8 kb ssDNA immune response strong mild expression short-lived long-lived (possible integration into host genome) diameter (nm) ~90 ~20 receptor coxsackievirus adenovirus receptor (CAR)/α5 integrin different (dependent on serotype) cargo (kb) up to 10 ~4.8 biosafety level (BSL) 2 1 (in absence of helper virus)

True “shuttle” vector?

The AdEasy system PacI-linearized DNA 293T cells Adenovirus The naming in this figure can be misleading. pAdEasy-1 is shuttling Between E. Coli and 293T, not the “Shuttle Vector”. PacI-linearized DNA 293T cells Adenovirus Figure from http://www.the-scientist.com/?articles.view/articleNo/13044/title/Delivering-the-Goods/

Bac-To-Bac® (LifeTechnologies, developed by Monsanto)

Fuzzy use of the term shuttle vector The term shuttle vector (used in the figure on the slide 12, which is from here) is used very loosely here as this vector cannot propagate in 293T cells. First, it has to recomine with the “true” shuttle vector, which is pAdEasy-1. However, the term shuttle vector is used loosely in the literature for both the E. coli vector, into which the gene of interest is cloned and for the vector that carries the actual replication signals for both E. coli and 293T cells (or in case of the Bac-to-Bac system insect cellls). Also the AdEasy manual used the term differently from the orthodox definition. The original publication of the Bac-to-Bac system in 1993 and the manual of the corresponding Invitrogen kit reserves the term for the large baculoviral genome which replicates in both insect cells and E. coli.

Recombinant Retroviruses Vesicular Stomatitis Virus envelope proteins Packaging signal

Recombinant Lentivirus Recombinant Lentiviruses Enveloped, ssRNA (= mRNA) genome Most commonly used recombinant lentivirus HIV-1-based Transduces deviding and non-dividing cells BSL-2 (some experiments require BSL-3) Cargo up to ~8 kb Services available from FUGU

Next meeting Different methods to to express two (or even more) genes from one vector Readme (IRES vs. 2A peptide): http://journals.plos.org/plosone/article?id=10.1371 /journal.pone.0018556 Exceptionally in room BM B136A (1st floor)

Miscellanies Plasmid 101 book from Addgene: http://info.addgene.org/download-addgenes-ebook-plasmids-101-2nd-edition