Transgenic approaches using I-SceI are powerful genome modification methods for creating heritable modifications in eukaryotic genomes. Such modifications are ideal for studying putative promoters and their temporal and spatial expression patterns in real time, in vivo. Central to this process is the initial engineering of a plasmid construct containing multiple DNA modules in a specific order prior to the integration into the target genome. One popular way of doing this is based upon the pGateway system, the modular form of which described in this chapter is known as pTransgenesis. We will initially describe the protocol of obtaining the plasmid construct containing the required sequence modules, and then the process of integrating the construct into the genome of a Xenopus embryo via co-injection with I-SceI and subsequent screening for transgenics.

The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes.
We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated.
Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.