C Pritchard

Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid

We have developed an X-irradiation:cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.

Hematopoietic Expression of Oncogenic BRAF Promotes Aberrant Growth of Monocyte-Lineage Cells Resistant to PLX4720

Mutational activation of BRAF leading to expression of the BRAFV600E oncoprotein was recently identified in a high percentage of specific hematopoietic neoplasms in monocyte/histiocyte and mature B-cell lineages. Although BRAFV600E is a driver oncoprotein and pharmacologic target in solid tumors such as melanoma, lung, and thyroid cancer, it remains unknown whether BRAFV600E is an appropriate therapeutic target in hematopoietic neoplasms. To address this critical question, we generated a mouse model expressing inducible BRAFV600E in the hematopoietic system, and evaluated the efficacy of pathway-targeted therapeutics against primary hematopoietic cells. In this model, BRAFV600E expression conferred cytokine-independent growth to monocyte/macrophage-lineage progenitors leading to aberrant in vivo and in vitro monocyte/macrophage expansion. Furthermore, transplantation of BRAFV600E-expressing bone marrow cells promoted an in vivo pathology most notable for monocytosis in hematopoietic tissues and visceral organs. In vitro analysis revealed that MAP–ERK kinase inhibition, but not RAF inhibition, effectively suppressed cytokine-independent clonal growth of monocyte/macrophage-lineage progenitors. However, combined RAF and phosphoinositide 3-kinase (PI3K) inhibition effectively inhibited cytokine-independent colony formation, suggesting autocrine PI3K pathway activation. Taken together, these results provide evidence that constitutively activated BRAFV600E drives aberrant proliferation of monocyte-lineage cells. Implications: This study supports the development of pathway-targeted therapeutics in the treatment of BRAFV600E-expressing hematopoietic neoplasms in the monocyte/histiocyte lineage. Mol Cancer Res; 11(12); 1530–41. ©2013 AACR.

Construction of Lambda Libraries from Large PFGE Fragments

Pulsed-field gel electrophoresis (PFGE) has the capacity to fractionate large fragments of DNA up to thousands of kilobases in size. This aspect of the technique has been exploited for constructing long-range restriction maps of chromosomes from many different species including humans (see Chapters 14 , 15 , and 18 ). Besides its use for analytical purposes, PFGE has also been used as a preparative tool. Intact DNA obtained from preparative PFGE gels has been used for cloning into yeast artificial chromosome (MC) vectors (see Chapter 16 ) and for constructing jumping libraries (1). In addition, DNAeluted from PFGE gels has been used for generating libraries with a smaller insert size (2-7). In this latter procedure, DNA from a somatic cell hybrid is digested with a rare-cutting restriction enzyme, separated by PFGE, and the DNA from a particular PFGE fragment is eluted, digested, and cloned into a plasmid or phage vector. The resulting library is then screened with a species-specific probe to identify DNA segments from the donor chromosome of the hybrid. This use of preparative PFGE has had widespread application in the cloning of DNA close to several important disease genes, namely cystic fibrosis (4,6), Duchenne muscular dystrophy (2), choroideremia (7), polycystic kidney disease (3), and Huntington disease (5).