Future studies will incorporate the use of well defined insulator elements in BAC linking vectors to assemble multi-reporter gene DNA constructs.
BACLink -GM and -SP linking vectors were generously provided by Claire Huxley [ 10]. pLD53-SCAEB recombination vector was generously provided by Shiaoching Gong [ 8] and the mini lamda vector was generously provided by Donald M. Court [ 19].
Trap homology Arm3 was cloned into both BACLinking vectors as the second common homology arm in addition to a region of the chloramphenicol acetyltransferase coding sequence that is present in the vector backbone of both linking vectors.
Past work has resulted in the generation of BAC linking vectors BACLink-GM, and BACLink-SP for the purpose of combining the components of one large gene, whose overlapping regions extended over multiple BAC clones, into one BAC vector [ 10].
This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together.
We reasoned that this strategy could also be used to link different genes of interest together, by engineering a second homology arm common to both BAC linking vectors (arm 3, Fig. 1A).
As proof of principle and in an effort to generate an animal model to mark multiple cell types relevant to bone biology, we have subcloned two genomic DNA fragments into BAC linking vectors GM and SP. One genomic DNA fragment contains the gene Acid Phosphatase 5 (Acp5), better known as Tartrate Resistant Acid Phosphatase (Trap), which is expressed in osteoclasts [ 11, 12].
Similarly, overlap extension PCR cloning also requires purification of the first round PCR products (vector and insert) and an additional round overlap extension PCR, which usually generates multiple bands, for producing linked vector and insert.
The coarse-grained model is constructed as a series of linked vectors following the contour of the polymer backbone.
We now describe a basic probablistic model for linking the vector π to the observed set of N mutations, as proposed by Durinck et al. (2011) for CNLOH events and generalized by Greenman et al. (2012).
This convex combination of link rate vectors allocates a time fraction of α k to every link vector v k to form the polyhedral rate region.
