genomics studies that have resulted from the availability of whole-genome sequences.
These novel cloning strategies rely onsite-specific DNA recombination techniquesand
significantly reduce the time and effort involved in generating recombinant DNA vectors
for gene analysis and cDNA library construction (cDNA is a DNA sequence that is comp-
lementary to the coding sequence of an RNA transcript). Three systems are currently avail-
able that work efficiently for large-scale cloning projects: GatewayTM (Invitrogen),
CreatorTM(Clonetech), and the Univector system (Liu et al. 1998), also known as the
EchoTMsystem (Invitrogen).
7.3.1.1. Gateway Cloning.The Gateway cloning system takes advantage of elements
that evolved naturally in the life cycle of the bacteriophage lambda(l). During this
cycle, the bacteriophage passes from a lysogenic phase, in which the viral genome is
stably incorporated into the host genome, to a lytic phase, in which the host cell ruptures
(lyses) and infectious phage particles are released [for a more recent review of lambda
development, see Oppenheim et al. (2005)] (Fig. 7.11).
The Gateway cloning system utilizes modifiedattrecombination sites, together with an
integration enzyme mix containing Integrase (Int) and Integration Host Factor (IHF) pro-
teins (BP clonase) and an excision/integration enzyme mix containing the Int, IHF, and
Figure 7.11.For lysogeny, the viral DNA is incorporated into the host genome by a process of
recombination between common sequences, theattsites, in the two genomes. Bacteriophagelcon-
tains anattPsite (Pfor phage), and the hostE. coliDNA contains anattBsite (Bfor bacterium). A
number of proteins are required for this recombination:l-derived Integrase (Int) andE. coli–derived
Integration Host Factor (IHF) allowlto enter the lysogenic phase of its life cycle, and IHF, Int, andl-
derived Excisionase (Xis) allowlto excise from theE. coligenome and enter the lytic phase of its
life cycle.
172 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION