scholarly journals The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

2008 ◽  
Vol 82 (16) ◽  
pp. 7818-7827 ◽  
Author(s):  
Filip Šenigl ◽  
Jiří Plachý ◽  
Jiří Hejnar
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Cpg Island ◽  
Cpg Islands ◽  
Transcriptional Silencing ◽  
Retroviral Vectors ◽  
Core Element ◽  
Promoter Sequences ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

scholarly journals Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

2010 ◽  
Vol 84 (13) ◽  
pp. 6626-6635 ◽  
Author(s):  
Julia D. Suerth ◽  
Tobias Maetzig ◽  
Melanie Galla ◽  
Christopher Baum ◽  
Axel Schambach
Keyword(s):  
Mammalian Cells ◽  
High Titer ◽  
Viral Proteins ◽  
Retroviral Vectors ◽  
Long Terminal Repeats ◽  
Rous Sarcoma ◽  
Vector Systems ◽  
Cellular Genes ◽  
Integration Sites ◽  
Transcriptional Elements

ABSTRACT Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

scholarly journals Methylation-Mediated Transcriptional Silencing in Euchromatin by Methyl-CpG Binding Protein MBD1 Isoforms

1999 ◽  
Vol 19 (9) ◽  
pp. 6415-6426 ◽  
Author(s):  
Naoyuki Fujita ◽  
Shin-ichiro Takebayashi ◽  
Katsuzumi Okumura ◽  
Shinichi Kudo ◽  
Tsutomu Chiba ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Cpg Islands ◽  
Transcriptional Silencing ◽  
Pericentromeric Region ◽  
Chromosome 1 ◽  
Reverse Transcription Pcr ◽  
Genome Methylation

ABSTRACT DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16,VHL, and E-cadherin and from an imprintedSNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested inDrosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

Analysis of Adhesion Plaque Structure and Function in the Mechanism of Transformation by Rous Sarcoma Virus

1982 ◽  
Vol 40 ◽  
pp. 110-113
Author(s):  
L.R. Rohrschneider ◽  
M.J. Rosok ◽  
L.E. Gentry
Keyword(s):  
Rous Sarcoma Virus ◽  
Mammalian Cells ◽  
Neoplastic Transformation ◽  
Structure And Function ◽  
Excellent Opportunity ◽  
Cells In Culture ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
And Function ◽  
Adhesion Plaque

Rous sarcoma virus (RSV) was originally isolated from a fibrosarcoma of a chicken. This virus also will efficiently infect and transform all avian cells in culture as well as most mammalian cells. The mechanism of transformation by RSV is therefore universal and this system offers an excellent opportunity to investigate the mechanism of neoplastic transformation.

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