Development of an in vitro system to determine the causes of aberrant, leukaemogenic V(D)J recombination reactions

Aims

This project aims to provide an in vitro model to replace the use of mice in research on DNA recombination during B and T cell development.

Background

DNA rearrangement is at the heart of the ability of B and T lymphocytes to generate a diverse array of immunoglobulins and antigen receptors to enable vertebrates to fight infections. Termed V(D)J recombination the process of breaking and rejoining segments of DNA occurs early during B and T cell development. It is inherently risky and up to 40% of lymphoid cancers appear to have arisen from aberrant V(D)J recombination.

Studies of V(D)J recombination rely on the use of the mouse and are hampered by the complexity of isolating cells undergoing recombination. Recently, a system has been developed which allows inducible recombination, this is however dependent on obtaining primary pro-B cells from transgenic mice. The goal of this research is to develop an immortalised cell line from the pro-B cells which would replace the use of around 10,000 mice a year.

Research details and methods

Pro-B cells will be immortalised using Abelson murine leukaemia virus, which is a well-established method of making cell lines from primary mouse B cells. The cell line will be compared with primary cells to test whether it undergoes recombination of the same magnitude and timing as the primary cells, and whether it retains all other epigenetic changes associated with recombination. The cell line will subsequently be used to better understand the safeguards that prevent chromosome translocations during V(D)J recombination.

V(D)J recombination generates a highly diverse repertoire of antigen receptor genes to allow vertebrates to fight a vast range of infections. However, since the reaction involves the breakage and rejoining of DNA, it is inherently risky and between 35-40% of lymphoid cancers appear to have arisen from mistakes in V(D)J and class switch recombination reactions. Since lymphoid cancers are the fourth most common cancer type, with 10,000 new cases per year in the UK alone, there is a strong need to better understand what triggers these aberrant recombination reactions. The experiments to investigate aberrant recombination reactions rely heavily on the use of mice and, until recently, have been hampered by the scarcity of cells undergoing recombination. V(D)J recombination occurs only in a fleeting population of cells during B and T cell development and, once it has occurred, the cells differentiate. Thus, few cells can be purified and it is very hard to isolate cells exactly at the point that they are undergoing V(D)J recombination. We have now developed a novel inducible V(D)J recombination system in which recombination can be activated at will. This is an extremely powerful new tool that has opened up many new avenues of research to examine how mistakes in V(D)J recombination trigger cancer. Currently this inducible V(D)J recombination system relies on isolating primary pro-B cells from transgenic mice. Thus, although many new projects are now possible, it is estimated that these projects would use 40,000‒50,000 mice over the next five years. Here, we propose to completely replace the use of these mice by generating a cell line from the pro-B cells. This cell line will be compared with primary cells to test if it undergoes recombination with the same magnitude and timing as the primary cells and if it retains all other epigenetic changes associated with recombination. If it does, then it will be an extremely powerful tool to address a plethora of questions about how aberrant recombination reactions are triggered. The final part of this studentship will examine one of these questions, namely to better understand the safeguards that prevent chromosome translocations during V(D)J recombination. These are a major cause of cancer and, to prevent translocations occurring, it is vital that the DNA regions undergoing recombination are activated coordinately and that these regions are brought together before double strand breaks are introduced. So far, nothing is known about the temporal regulation of these processes but the inducible V(D)J recombination system makes these experiments possible for the first time. Thus, this work could provide significant insights into the safeguards that suppress leukaemogenic translocations. Together, these studies aim to generate an extremely powerful new tool that is likely to be of great utility not only to our lab but also to other labs throughout the world with related interests in V(D)J recombination. This cell line will enable numerous new projects to determine how aberrant recombination reactions lead to lymphoid cancers and will result in the complete replacement of substantial numbers of mice.