Construction of a high-performance human fetal liver-derived lentiviral cDNA library

Abstract

The gene transduction method is a very powerful tool, not only in basic science but also in clinical medicine. Regenerative medicine is one field that has close connection with both basic and clinical. Recently, it has been reported that induced pluripotent stem (iPS) cells can be produced from somatic cells by a three or four gene transduction. We have also recently reported that lentiviral gene transfer of the tal1/scl gene can efficiently differentiate non-human primate common marmoset ES cells into hematopoietic cells without the support of stromal cells. In this study, we constructed a high-performance human fetal liver-derived lentiviral expression library, which contains a high number of individual clones, in order to develop a very helpful tool for understanding early hematopoiesis and/or hepatocytosis for future regenerative medicine. Our lentiviral cDNA library consisted of more than 8 x 10(7) individual clones, and their average insert size was >2 kb. DNA sequence analysis for each individual inserted cDNAs revealed that >60% contained the full-length protein-coding regions for many genes including cytokine receptors, cytoplasmic proteins, protein inhibitors, and nuclear factors. The transduction efficiency on 293T cells was 100% and the average size of an integrated cDNA was ~1.1 kb. These results suggest that our lentiviral human fetal liver cDNA expression library could be a very helpful tool for accelerating the discovery of novel genes that are involved in early hematopoiesis and hepatopoiesis and to make the use of iPS cells more efficient in the field of regenerative medicine.

Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells

“…The TAL1 cDNA was then transferred to the CSII-EF-RfA-IRES-Puro r plasmid using Gateway LR clonase (Invitrogen) to produce CSII-EF-TAL1-IRES-Puro r . The vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped lentiviral vector preparation was performed as described previously [10] with the exception that Fugene HD (Roche, Mannheim, Germany) was used for transfection instead of polyethyleneimine. Human iPS cell lines were transduced with CSII-EF-TAL1-IRES-Puro r lentiviral vector in the presence of polybrene (8 µg/ml; Sigma) and iPS cells expressing TAL1 were selected using puromycin (2 µg/ml; InvivoGen, San Diego, CA, USA).…”

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells

“…The TAL1 cDNA was then transferred to the CSII-EF-RfA-IRES-Puro r plasmid using Gateway LR clonase (Invitrogen) to produce CSII-EF-TAL1-IRES-Puro r . The vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped lentiviral vector preparation was performed as described previously [10] with the exception that Fugene HD (Roche, Mannheim, Germany) was used for transfection instead of polyethyleneimine. Human iPS cell lines were transduced with CSII-EF-TAL1-IRESPuro r lentiviral vector in the presence of polybrene (8 mg/ml; Sigma) and iPS cells expressing TAL1 were selected using puromycin (2 mg/ml; InvivoGen, San Diego, CA, USA).…”

APOA-1 is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans

“…The previously generated human fetal liver-derived Entry cDNA library [12] was used in this study. Briefly, 34 μg (1-2×10 5 cDNA clones) of the library was mixed with 20 μg of pCAG-HIVg/p and 20 μg of pCMV-VSVG-RSV-Rev as the packaging plasmids in 3.5 ml of FBS-free DMEM, and then 370 μl of 1 mg/ml polyethylenimine (PEI) was added to the mixture.…”

“…In addition, we constructed a high-performance human fetal liver (FL)-derived cDNA lentiviral library as a tool to facilitate the discovery of novel genes that are involved in the expansion of HSCs, erythropoiesis and/or liver development [12]. During embryogenesis, the FL is the major site of hematopoiesis, particularly erythropoiesis.…”