Each of pCDNA3.1-GFP, pCEP4-GFP, pIg-GFP and pIE-GFP plasmids was transfected into H1299-AID-15 cells separately. therapy and diagnosis. However, oftentimes, antibodies obtained from hybridoma or antibody libraries exhibit low affinity [1,2], which is usually insufficient for use in diagnosis and medical treatment. To this end, numerous antibody display technologies have been developed for antibody affinity maturation, such as phage display [3,4], bacteria display [5,6,7], yeast display [8,9], and mammalian cell display [10,11,12,13]. Among all these techniques, the mammalian cell display platform has the advantages of protein expression, more advanced peptide folding and post-translational modifications [14,15,16]. To generate an antibody library in mammalian cells, three main steps are required. 1. Cloning the antibody mutants into mammalian cell expression vectors to generate an antibody library in bacteria. 2. Purifying the library plasmids and transfect the plasmids into mammalian cells. 3. Antibiotic selection to generate stable cells with antibodies displayed around the cell surface. You will find two kinds of mammalian cell expression vectors (chromosome-integrating [17] and episomal vectors [10,16,18]) which can be used to develop libraries. In chromosome-integrating vector systems, cells without plasmids inserted into their genome will be killed by antibiotics during the antibiotic selection process. Due to the low efficiency of genome integration (i.e., 1%) [19], only small size libraries (106range) can be generated using chromosome-integrating plasmids in mammalian cells. In the system using episomal vectors, the integration of the antibody-expression vectors into cell genome is not required. However, low transfection efficiency should be chosen to generate cells with low copy number plasmids for expression within the cell [16,20,21,22]. Much like chromosome-integrating vectors, antibiotic selection also needed to generate stable library cells, and most of the cells will be killed by the antibiotic. The antibody libraries generated by episomal vectors are also small. Activation-induced cytidine deaminase (AID) enzyme deaminates cytidine residues in IgG genes to initiate somatic hypermutation in both B cells and non-B cells [12,23]. It has been used to generate antibody libraries K-Ras G12C-IN-2 in mammalian cells [13,16,23,24]. Coupling mammalian cell display and AID-induced somatic hypermutation (SHM), several groups have developed their own antibody selection [25,26] and affinity maturation systems [10,12,13,16,27,28]. However, the rate of AID-induced SHM is usually less than105bp per generation in vitro [12,23], and is over 100-fold lower than the centroblast stage K-Ras G12C-IN-2 of B cell differentiation (103bp per generation) [16]. Thus, improving the rate of AID-induced SHM in vitro has great value in mammalian cell display technologies. It has been previously reported that AID could mutate the target antibody gene encoded in an episomal vector pCEP4 (Invitrogen, K-Ras G12C-IN-2 Grand Island, NY, USA) in mammalian cells, and significant antibody affinity improvement was achieved in these pCEP4 vector-based systems [16,29]. Unlike regular vectors, pCEP4 can stably replicate and be K-Ras G12C-IN-2 managed within mammalian cells as episomes outside of chromosomes. This confers a higher level of transcription of the desired antibody gene than integrated vectors [13,30]. Higher level transcription of the interested antibody gene can generate a higher AID-induced mutation rate [31]. It is widely known that enhancer (a short (501500 bp) region of DNA) can bind with specific transcription factors to enhance the transcription of nearby genes [32,33]. Thus, increasing the frequency of AID-induced SHM to nearby transcribed genes [34,35,36,37,38]. We hypothesize that, by placing an enhancer in the pCEP4 vector, higher degree of AID-induced SHM to the target gene on pCEP4 should be obtained. In this work, two different enhancers which were identified from your major introns of the mouse immunoglobulin heavy (IgH) loci (Ig enhancer) [39] and x light chain loci (Ek enhancer) [40] were chosen for the experiments. We tested the mutation rates of a targeted GFP gene sequence in pCEP4 vectors made up of one or two different enhancers. The plasmid with two enhancers experienced two-fold AID-induced SHM than the initial pCEP4 vector. Furthermore, we used this system to mature an antibody against high mobility group box 1 (HMGB1) protein [41,42,43,44,45]. Here we chose to mature a Rabbit Polyclonal to GPR108 full-length anti-HMGB1 antibody (3B1) [46]. Using the designed.