Human molecular Genetics
Instituto de Salud Carlos III (ISC III)

Introduction: The immune system recognizes antigens (Ag) and  pathogens through different mechanisms, including specific B and T lymphocytes. In the case of B lymphocytes upon antigen encounter these are activated and differentiate into plasma cells, and secretion of specific immunoglobulins (Igs) occur. In recent years, technologies have been developed for determining at the molecular level the different specificities of the Igs expressed in B lymphocytes. This allows to define the extent and variability of Igs repertoires each case. Also, it is posible to identify at the molecular level and to isolate the variable regions, for possible subsequent expression. Such approaches are highly innovative and their application to the field of infectious diseases will define those IgS able to recognize the pathogens efficiently.
Goals: Our group is performing the study of the  gene repertoiresof  heavy chain variable region of IgS (VH) in mouse and human. It is required to complement these analyses with those corresponding to the light chain variable genes (VL). To do that, we propose:
1. To select the specific conditions for the analysis of genes of VL (K and L chains) expressed in basal conditions of homeostasis in human and mice.
2. Using the murine model, to analyze the VL repertoires that are used after immunization protocols with bacterial lipopolysaccharide (LPS,  recognized by the receptor TLR4 ), capable of inducing a polyclonal activation of B lymphocytes.
3. To check the relationship of this response via TLR4 using genetically modified animals in which the expression of TLR4 or MyD88 (MyD88 and TLR4-KO-KO) molecule has been abrogated.

Main techniques used:
- Dissection and isolation and purification of cell suspensions of B cells by flow cytometry or immuno-magnetic techniques.
- Preparation of mRNA, amplification of light chain transcripts (RT-PCR), cloning and sequencing, massive sequencing.
- Analysis of sequence data: alignment, analysis of variability of the joint (joint-region) of VL regions, mutation analysis and antigen selection analysis. Phylogenetic trees.

Molecular Biomedicine
Isabel Cortegano Jimeno