Biomedical Engineering Reference
In-Depth Information
Table 2
Types of Entry clone/Destination vector combinations for 2-, 3-, and 4-fragment cloning
Fragments
Entry 1
Entry 2
Entry 3
Entry 4
Destination vector
2
attL
4-
attR
1
attL
1-
attL
2
attR
4-
attR
2
3
attL
4-
attR
1
attL
1-
attL
2
attR
2-
attL
3
attR
4-
attR
3
3
attL
4-
attL
5
attR
5-
attR
1
attL
1-
attL
2
attR
4-
attR
2
4
attL
4-
attL
5
attR
5-
attR
1
attL
1-
attL
2
attR
2-
attL
3
attR
4-
attR
3
Table 3
Examples of some styles of combinatorial construct designs
Type of construct
First entry clone
Second entry clone
Third entry clone
Basic protein
expression
Promoter
(
attL
4-
attR
1)
ORF (
attL
1-
attL
2)
Tagged protein
expression
Promoter
(
attL
4-
attL
5)
N-terminal fusion
(
attR
5-
attR
1)
ORF (
attL
1-
attL
2)
Tagged protein
expression
Promoter
(
attL
4-
attR
1)
ORF (
attL
1-
attL
2)
C-terminal fusion
(
attR
2-
attL
3)
Gene expression
with reporter
Promoter
(
attL
4-
attR
1)
ORF (
attL
1-
attL
2)
IRES-GFP (
attR
2-
attL
3)
shRNA expression
Promoter
(
attL
4-
attR
1)
GFP (
attL
1-
attL
2)
shRNA (
attR
2-
attL
3)
designed with a specifi c
att
site confi guration to permit maximum
fl exibility in combinatorial construction. While several commercial
fl avors of Multisite Gateway are available, the majority of them are
designed to utilize commercially available Destination vectors
(
attR
1-
attR
2) rather than commercially available ORF Entry
clones (
attL
1-
attL
2). As there are tens of thousands of these Entry
clones available and only a few hundred Destination vectors, this
system is not nearly as universally applicable. Instead, we and oth-
ers have focused systems that require the construction of new
Destination vectors, but can take full advantage of the already
available standard Gateway Entry clones [
4
-
7
]. This system per-
mits construction of clones with 2, 3, or 4 Entry clone fragments
in multiple confi gurations as shown in Table
2
.
In this format, only two different Destination vectors are
required:
attR
4-
attR
2 for 2- or 3-fragment cloning, and
attR4-
attR3
for 3- or 4-fragment cloning. The choice of which 3-fragment
pathway to use likely will depend on your desire to include stan-
dard
attL
1-
attL
2 Entry clones. Table
3
shows some examples of
how these fragments can be used to make expression vectors in the
