In vivo expression of GFP transgene delivered via a replicating feline leukemia virus

Pan, Judong; Aldubaib, Musaad; Liao, Chun-Peng; Fujino, Yasuhito; Hinton, David R.; Hayes, Kathleen; Mathes, Lawrence E.; Roy-Burman, Pradip

doi:10.1016/j.vetmic.2005.07.015

Cited by 6 publications

(5 citation statements)

References 37 publications

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“…One microlitre of product from the first round of amplification was used in the second round of PCR with subgroup specific primers as follows: RB59 and RB17 (specific for FeLV-A) that generate a 1072 bp amplicon (Chen et al, 1998), RB53 and RB17 (specific for FeLV-B) generating a 866 bp product (Pan et al, 2005) and RB58 and RB47 (specific for FeLV-C) that yield a 1755 bp amplicon (Mathes et al, 1994). We did not test samples using FeLV-T-specific primers.…”

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confidence: 99%

Naturally occurring feline leukemia virus subgroup A and B infections in urban domestic cats

Coelho

Bomfim

Caxito

et al. 2008

Journal of General Virology

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A nested-PCR (n-PCR) was used to detect feline leukemia virus (FeLV) proviral DNA in blood samples from 464 sick and 608 healthy domestic cats (Felis catus) selected by convenience, and a significantly high prevalence of FeLV infection was observed. n-PCR results revealed the presence of FeLV proviral DNA in 47.2 % of sick cats and 47.4 % of healthy cats. Phylogenetic analysis revealed that FeLV samples from healthy or sick cats were grouped into separate clades. We determined FeLV subgroups by an n-PCR based on the envelope (env) gene. The partial env gene of FeLV Minas Gerais (MG) samples were compared to various exogenous FeLV isolates and endogenous (enFeLV) provirus from the same region. FeLV-B MG samples were more similar to endogenous sequences and to natural FeLV-B isolates than to either FeLV-A or FeLV-C. The results revealed the circulation of FeLV-B in large populations of urban domestic cats in Brazil.Feline leukemia virus (FeLV) is an exogenous retrovirus, belonging to the genus Gammaretrovirus, which infects domestic and sporadically wild cats (Daniels et al., 1999; Arjona et al., 2007). By comparison of the envelope (env) sequence variation it has been possible to identify four FeLV subgroups: FeLV-A, -B, -C and -T, each of which uses distinct cellular receptors to initiate infection of the host cell (Boomer et al., 1997;Tailor et al., 1999; Anderson et al., 2001;Mendoza et al., 2006;Cheng et al., 2007). FeLV has been associated with fatal neoplasia, degenerative diseases of the haematopoietic system and immunodeficiency (Chandhasin et al., 2004;Hofmann-Lehmann et al., 2006;Collado et al., 2007).Traditionally, FeLV infections are diagnosed by virus isolation (VI), immunofluorescent antibody tests (IFA) or ELISA for the detection of soluble antigens (usually the p27 core protein) in whole blood, serum or plasma (Levy et al., 2008). However, FeLV infections in cats with a weakened immune response are not reliably detected by serological methods . Recently, molecular methods such as PCR assays have been described for the use of detection of FeLV provirus DNA in peripheral blood mononuclear cells (PBMC), allowing virus detection independently of antibodies presence or viraemia, and enabling detection in latently infected cats (Hofmann-Lehmann et al., 2001;Torres et al., 2005;Tandon et al., 2008). Moreover, a highly sensitive nested-PCR (n-PCR) assay was developed and it is able to distinguish between endogenous and exogenous FeLV by using inner and outer pairs of primers based on the U3 long terminal repeat (LTR) region of FeLV provirus (Miyazawa & Jarrett, 1997 Veterinarians were requested to take EDTA-blood samples (1.0 ml) from the cats regardless of their clinical status. Written consent was obtained from each cat owner, and ethical guidelines were always followed.Statistical analyses were performed using the x 2 or Fisher's exact tests using the GraphPad InStat version 3.05 for Windows (GraphPad Software). Sample prevalence estimates were calculated and reported as the per cent of cats with ...

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confidence: 99%

Naturally occurring feline leukemia virus subgroup A and B infections in urban domestic cats

Coelho

Bomfim

Caxito

et al. 2008

Journal of General Virology

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“…(A) Representative fluorescent microscope pictures of 10 m liver sections from: (1) normal animal not injected with enhanced green fluorescent protein-vascular endothelial cells (EGFP-VEC) ( n = 5); (2) normal animal injected with EGFP-VEC ( n = 5); and (36)four metastases-bearing animals that received EGFP-VEC. (B) Correlation of manual enumeration and qPCR.…”

Section: Resultsmentioning

confidence: 99%

“…A general review article described genes that could be detected in tissue by qPCR after adoptive transfer (6), but transgene analysis and computations were not fully described and tissue analysis was not presented. This method has been used successfully to evaluate fetal microchimerism in wild-type female mice bred with EGFP-transgenic males (4,5) and critical calculations for quantification of EGFP transgene were well described in a feline viral infection model (2). However, based on the low frequency with which these few methodology publications have been cited, and the paucity of published articles using qPCR to quantitate biolocalized cells, it appears that this important methodology has failed to reach most immunologists and cell biologists conducting adoptive transfer experiments.…”

Section: Introductionmentioning

confidence: 99%

Real-Time PCR to Determine Transgene Copy Number and to Quantitate the Biolocalization of Adoptively Transferred Cells from EGFP-Transgenic Mice

Joshi

Pittman²,

Haisch³

et al. 2008

BioTechniques

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Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

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“…[1][2][3] Successful amplification of fulllength HIV-1 envelope genes has been previously described using nested PCR from the DNA of patient PBMC [12][13][14][15][16] and by RT-PCR from plasma viral RNA. [8][9][10][11] Both of these methods needed nested PCR, which not only increased the probability of mutation, but also amplfied full-length env hardly. As HIV mutates rapidly in vivo, it is important to identify the true in vivo envelope sequence to study HIV membrane properties.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3] PCR methods for the amplification of fulllength HIV-1 envelope genes from primary and continuous cell cultures have been previously described. 1,2,[4][5][6][7] The amplification of full-length envelope genes from plasma of HIV patients using RT-PCR [8][9][10][11] or from peripheral blood mononuclear cells (PBMC) DNA extracts of HIV patients requires the use of nested primer polymerase chain reactions (PCR). [12][13][14][15][16] These methods have several disadvantages.…”

Section: Introductionmentioning

confidence: 99%

Amplification of Complete HIV-1 Envelope Genes from Purified CD4+ T Cell Populations

Jiao¹,

Li²,

Wei³

et al. 2009

N A J Med Sci

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Polymerase chain reaction (PCR) amplification of fulllength HIV-1 envelope genes directly from uncultured clinical samples or from plasma of HIV patients using RT-PCR or from peripheral blood mononuclear cells (PBMC) DNA extracts of HIV patients is difficult. As HIV mainly infected CD4 positive cells, this paper first describes a sensitive method for the amplification of fulllength HIV-1 envelope genes from purified CD4+ cells, and compares this method with other, less effective, procedures. This method were suitable for amplification of HIV-1 envelope genes directly from clinical samples especially from those of low viral load.

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In vivo expression of GFP transgene delivered via a replicating feline leukemia virus

Cited by 6 publications

References 37 publications

Naturally occurring feline leukemia virus subgroup A and B infections in urban domestic cats

Naturally occurring feline leukemia virus subgroup A and B infections in urban domestic cats

Real-Time PCR to Determine Transgene Copy Number and to Quantitate the Biolocalization of Adoptively Transferred Cells from EGFP-Transgenic Mice

Amplification of Complete HIV-1 Envelope Genes from Purified CD4+ T Cell Populations

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