Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins

Abstract

The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

First landscape of binding to chromosomes for a domesticated mariner transposase in the human genome: diversity of genomic targets of SETMAR isoforms in two colorectal cell lines

“…Here, we derived the DNA binding landscape of several SETMAR isoforms in the colorectal cell lines SW48 and HT29. These lines display specific expression profiles of SETMAR isoforms: HT29 cells express a single SETMAR isoform (V2) whereas SW48 cells express several (V1, V2, X2, V5 and HSMAR1) [4]. We also revisit the annotation of the human genome for the Hsmar1 element and its associated 80-pb miniature element, MADE1, using logol [23] and BLAST+, and we could detect and annotate ~2500 novel MADE1 copies.…”

“…HeLa cells, human colorectal cancer cell lineages (SW48, SW403, HT29), human melanoma cell lineages (BRIS, SK-MEL28and 518-A2), and human breast cancer cell lineages (MCF7, MDA-MB231, SKBR3 and T47D) were all cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), at 37 °C and with 5 % CO 2 . HeLa cell transfection with 1 µg of pVAX-HSMAR1 DNA was monitored as described [4].…”

“…Murine pre-immune and anti-HSMAR1 polyclonal sera were produced by DNA vaccination using ICANtibodies TM technology (In Cell Art, Nantes, France) as described [4]. Polyclonal antibodies (pA) contained in pre-immune and anti-HSMAR1 murine sera were purified using Protein A/G MagBeads (GenScript, Piscataway, USA).…”

“…Protein extracts from cultured cells, PAGE, transfer onto a polyvinylidene difluoride (PVDF) membrane, antibodies incubations and imaging with a FUJI LAS4000 imager were carried out as described [4].…”

“…Other variants resulting from single nucleotide polymorphisms can be found in human populations ( [3] and http://databases.lovd.nl/whole_genome/view/SETMAR). The detection of SETMAR in various colorectal [4], melanoma, breast cancer (Supplementary Figure S1), and leukaemia cell lines [5] confirmed that only five transcript variants were translated into protein isoforms (Figure 1, V1, V2, X2, V5 and an isoform with a molecular weight (MW) similar to that of HSMAR1), although differences exists from one cell line to another [4]. To our knowledge, it is not established which protein isoforms are present in healthy and cancerous tissues of patients.…”