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Notch signals regulate multiple cell fate decisions during metazoan development. During hematopoiesis, Notch affects both hematopoietic stem cells and committed progenitors. In hematopoietic stem cells, Notch signaling has the propensity to expand the stem cells, promote their self-renewal, and influence their survival. In committed progenitors, Notch signaling plays a key role in determining lymphoid cell fates. This review focuses on recent developments to understand the role of Notch signaling in early events in hematopoiesis.

Rapid developments have occurred both in laboratory medicine and in therapeutic interventions for the management of patients with chronic myelogenous leukemia (CML). With a wide array of laboratory tests available, selecting the appropriate test for a specific diagnostic or therapeutic setting has become increasingly difficult. In this review, we first discuss, from the point of view of laboratory medicine, the advantages and disadvantages of several commonly used laboratory assays, including cytogenetics, fluorescence in situ hybridization (FISH), and qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We then discuss, from the point of view of clinical care, the test(s) of choice for the most common clinical scenarios, including diagnosis and monitoring of the therapeutic response and minimal residual disease in patients treated with different therapies. The purpose of this review is to help clinicians and laboratory physicians select appropriate tests for the diagnosis and monitoring of CML, with the ultimate goal of improving the cost-effective usage of clinical laboratories and improving patient care.

Generation of high-titer retrovirus by transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high-titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient transfection has also increased the versatility of retrovirus-mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus-transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293-based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential transfection or cotransfection. Virus produced by transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.

Helper virus is a replication-competent virus that is sometimes present in stocks of replication-incompetent virus. There are several types of applications in which the presence of helper virus can be problematic. If animal infections are being done, helper virus can lead to leukemia, particularly if the infection is carried out pre- or neonatally. If retroviruses are being used for lineage analysis, helper virus may cause horizontal spread of the marker virus, creating false lineage relationships. This unit describes protocols for the detection of helper virus by a selectable marker assay, by rescue of an integrated provirus, or by measuring reverse transcriptase activity.

There are many applications in which retrovirus vectors are used as transduction agents. In some cases, the vector carries a gene that one wishes to express in a target cell in order to study the function of that gene. In other cases, the virus is used to introduce a histochemical marker gene into cells in order to follow their fate. Retrovirus vectors can also be used in a variety of cells type to investigate regulatory sequences in which a reporter gene and regulatory sequences are carried by the vector and to immortalize or transform primary cells by transduction of oncogenes. For each application, the infection protocol may vary and must often be optimized. Guidelines for infection of cells in some typical in vivo and in vitro experiments are presented in this overview.

A retrovirus vector is an infectious virus used to introduce a nonviral gene into mitotic cells in vivo or in vitro. The efficient and precise integration machinery of naturally occurring retroviruses is utilized to produce either a single copy or a few copies of the viral genome stably integrated into a host chromosome. This unit presents an overview of the retrovirus life cycle and a description of vector designs and packaging cell lines.

The steady-state level of c-myc proto-oncogene mRNA was investigated in the EBV-negative human B-lymphoma line BJAB and 2 sublines that have been converted by EBV into stable EBV-genome-carrying and EBNA-positive status. The EBV-converted sublines expressed c-myc at a 2- to 6-fold higher level than the original BJAB during exponential growth. The EBV-positive BJAB lines are known to differ from the parent line in several phenotypic characteristics, including increased agarose clonability, lower serum requirement and, in one case, increased tumorigenicity in nude mice. The pattern of increased c-myc expression accompanying EBV conversion was not observed in the myc/Ig translocation-carrying Burkitt lymphoma line RAMOS or in 2 of its EBV-converted sublines.