Plaque assay techniques for murine leukemia viruses

“…Virus-containing medium was screened for replicationcompetent ecotropic virus by using the XC plaque assay (32,37) and for amphotropic virus by using the S'L-assay (9,32 (28,48).…”

mentioning

confidence: 99%

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

McIvor¹,

Johnson²,

Miller³

et al. 1987

Mol. Cell. Biol.

View full text Add to dashboard Cite

Cell lines were established which produced high titers (_ 106 infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.Purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) are enzymes of purine metabolism, the absence of which in humans results in immunodeficiency disease. A severe combined immunodeficiency is associated with the lack of ADA activity (13), and a T-cell immunodeficiency is associated with the lack of PNP activity (12). Both conditions are recessive and autosomally inherited, and afflicted individuals suffer from opportunistic infections that lead to death early in childhood. The metabolic basis of these diseases has been studied intensively (see references 22 and 27 for reviews). Although a number of hypotheses have been proposed to explain the specific toxicity of these conditions to immune cells, there is much evidence that in the absence of one of these enzyme activities, deoxynucleoside substrates accumulate, resulting in unbalanced deoxynucleoside triphosphate pools, feedback inhibition of ribonucleotide reductase, and limited DNA synthesis which does not support an immunoproliferative response (22,27,46).The availability of cloned ADA and PNP coding sequences (14, 48) and progress in gene transfer technology (3,52) have led to the suggestion that the ADA-and PNPassociated immunodeficiencies might be reasonable candidates for initial attempts at human gene therapy (1,33). To * Corresponding author.

show abstract

“…The supernatant was spun again for 30 min at 16 300 g. This acellular viral extract was stored in liquid nitrogen. XC plaque assay was used to quantify the viral particles [16]. The viral preparation contained 10 3 particle forming units (PFU) of ecotropic virus/ml.…”

Section: Methodsmentioning

confidence: 99%

Peyer's Patches in Murine AIDS: Dissociation Between Lymphoproliferation and Anergy

Colombi¹,

Moutschen

Leval³

et al. 1997

Scand J Immunol

View full text Add to dashboard Cite

RadLV-Rs infection induces a murine immunodeficiency syndrome associated with a dramatic enlargement of spleen and lymph nodes. Surprisingly, the lymphoproliferation excludes thymus and Peyer's patches (PP). To understand the cellular interactions underlying lymphoproliferation further, the authors investigated the fate of PP in RadLV-Rs infected mice. The atrophy of PP was mostly due to the depletion of B cells, while the proportion of CD4 + and CD8 + T cells was increased. Nevertheless, B cell phenotype was modified with the emergence of lymphocytes with a low expression of B220 in infected PP. T cells characterized by a memory/ activated phenotype in control PP did not undergo phenotypical changes after viral infection (i.e. regarding Thy-1 and CD44 expression). Despite the absence of lymphoproliferation, PP T and B cells displayed altered responses to mitogens in vitro. Finally, alterations of the expression of adhesion molecules and vascular addressins could not explain the atrophy of PP by a reduced homing to this lymphoid site. B cells and T cells from normal PP are clearly different from lymph nodes (LN) lymphocytes. The authors propose that the particular functional state which characterizes PP lymphocytes influences the B cell/T cell crosstalk necessary for RadLV-Rs-induced lymphoproliferation.

show abstract

Plaque assay techniques for murine leukemia viruses

Cited by 1,253 publications

References 4 publications

A new class of murine leukemia virus associated with development of spontaneous lymphomas.

A new class of murine leukemia virus associated with development of spontaneous lymphomas.

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

Peyer's Patches in Murine AIDS: Dissociation Between Lymphoproliferation and Anergy

Contact Info

Product

Resources

About