1. 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

2. 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

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

4. 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)

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

6. 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):

7. 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

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

9. “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.

10. 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)

11. True “shuttle” vector?

12. 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

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

14. 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.

15. Recombinant Retroviruses
Vesicular Stomatitis
Virus envelope proteins
Packaging signal

16. 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

17. Next meeting
Different methods to to express two (or even more) genes from one vector
Readme (IRES vs. 2A peptide): /journal.pone
Exceptionally in room BM B136A (1st floor)

18. Miscellanies Plasmid 101 book from Addgene: