Generation of Human Induced Pluripotent Stem Cells Bearing an Anti-HIV Transgene by a Lentiviral Vector Carrying an Internal Murine Leukemia Virus Promoter

Previously, the Chen laboratory described the generation of a chimeric vector containing a murine leukemia virus (MLV) promoter internal to a lentiviral vector back bone. In this report, the authors report that this chimeric MLV/lentiviral vector can be used to generate induced pluripotent stem (iPS) cells. Kamata et al. demonstrate that these iPS cells are virtually indistinguishable from human embryonic stem cells and are capable of differentiating into several lineages

Human embyronic/atrial myosin alkali light chain gene. Characterization, sequence, and chromosomal location

We have isolated and sequenced the gene encoding the human embryonic/atrial myosin alkali light chain isoform (MLC-1emb/A). The gene is split into seven exons by six introns. The last exon, as in all MLC isoform genes sequenced to date, is completely 3'untranslated sequence. Comparison of the MLC-1emb/A isoform gene with the other MLC-1 genes showed that the exon-intron arrangement of the human MLC-1emb/A isoform gene is analogous to that of the other MLC-1 type isoform genes. We have also mapped the human MLC-1emb/a isoform gene to the long arm of chromosome 17. The corresponding mouse gene has been mapped to chromosome 11. This gene, together with a number of others such as the collagen alpha 1, galactokinase, and thymidine kinase genes, is part of the largest syntenic group between mouse and man

The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene fro

MicroRNA 142-3p Attenuates Spread of Replicating Retroviral Vector in Hematopoietic Lineage-Derived Cells While Maintaining an Antiviral Immune Response

We are developing a retroviral replicating vector (RRV) encoding cytosine deaminase as an anticancer agent for gliomas. Despite its demonstrated natural selectivity for tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific microRNA target sequence in RRV further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of microRNA 142-3p were inserted into the 3′ untranslated region of the RRV genome expressing the transgene encoding green fluorescent protein (GFP). Viral spread and GFP expression of these vectors in hematopoietic lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR, and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated peripheral blood mononuclear cells, vectors carrying the 142-3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse tumor model, RRVs with and without the 142-3pT sequences spread equally well in tumor cells; were strongly repressed in blood, bone marrow, and spleen; and generated antiviral immune responses. In an immune-deficient mouse model, RRVs with 142-3pT sequences were strongly repressed in blood, bone marrow, and spleen compared with unmodified RRV. Tissue-specific microRNA-based selective attenuation of RRV replication can maintain antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications

Study on the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell lines

Objective: Detecting the expression and mutation of human telomeric repeat binding factor (hTRF1) in 10 malignant hematopoietic cell line cells on the base of determining its genomic structure and its four pseudogenes to clarify if hTRF1 mutation is one of the factors of the activation of telomerase. Methods: hTRF1cDNA sequences were obtained from GenBank, its genome structure and pseudogenes were forecasted by BLAST and other biology information programs and then testified by sequencing. Real-time RT-PCR was used to detect the expression of hTRF1mRNA in 10 cell line cells, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-1, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji. Telomerase activities of cells were detected by using telomeric repeat amplification (TRAP)-ELISA protocol. PCR and sequencing were used to detect mutation of each exon of hTRF1 in 10 cell line cells. Results: hTRF1 gene, mapped to 8q13, was divided into 10 exons and spans 38.6 kb. Four processed pseudogenes of hTRF1 located on chromosome 13, 18, 21 and X respectively, was named as ΨhTRF1-13, ΨhTRF1-18, ΨhTRF1-21 and ΨhTRF1-X respectively. All cell line cells showed positive telomerase activity. The expression of hTRF1 was significantly lower in malignant hematopoietic cell lines cells (0.0338, 0.0108~0.0749) than in normal mononuclear cells (0.0493, 0.0369~0.128) (P=0.004). But no significant mutation was found in all exons of hTRF1 in 10 cell line cells. Four variants were found in part of intron 1, 2 and 8 of hTRF1. Their infection on gene function is unknown and needs further studies. Conclusion: hTRF1 mutation is probably not one of the main factors for telomerase activation in malignant hematopoietic disease