Position-independent human β-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken β-globin insulator

Takahito Inoue, Haruyoshi Yamaza, Yasuyoshi Sakai, Shinichi Mizuno, Ohno Mizuki, Naotaka Hamasaki, Yasuyuki Fukumaki

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Abstract

The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken β- globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I- hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human β-globin locus control region (LCR), the human β-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neo(R)) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken β-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human β-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse β(maj)-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human β-globin gene ranged from 51.6% to 765.6% of that in the mouse β(maj)globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human β-globin gene ranged from 52.8% to 58.3% of that in the mouse β(maj)globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.

Original languageEnglish
Pages (from-to)152-162
Number of pages11
JournalJournal of Human Genetics
Volume44
Issue number3
DOIs
Publication statusPublished - Jan 1 1999

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Dependovirus
Globins
Chickens
Gene Expression
Genes
Clone Cells
Transgenes
Genome
Neomycin
Genetic Therapy
Chromosomes
Locus Control Region
Viruses
Leukemia, Erythroblastic, Acute
Thymidine Kinase
Deoxyribonuclease I
Southern Blotting
Reverse Transcriptase Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)

Cite this

Position-independent human β-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken β-globin insulator. / Inoue, Takahito; Yamaza, Haruyoshi; Sakai, Yasuyoshi; Mizuno, Shinichi; Mizuki, Ohno; Hamasaki, Naotaka; Fukumaki, Yasuyuki.

In: Journal of Human Genetics, Vol. 44, No. 3, 01.01.1999, p. 152-162.

Research output: Contribution to journalArticle

Inoue, Takahito ; Yamaza, Haruyoshi ; Sakai, Yasuyoshi ; Mizuno, Shinichi ; Mizuki, Ohno ; Hamasaki, Naotaka ; Fukumaki, Yasuyuki. / Position-independent human β-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken β-globin insulator. In: Journal of Human Genetics. 1999 ; Vol. 44, No. 3. pp. 152-162.
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