Clonal diversity of vector-marked human henutopoietk progenitors recovered from longterm engrafted immune deficient mice

M. A. Pao, X. J. Yu, D. B. Kohn, Jan Nolta

Research output: Contribution to journalArticle

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Abstract

Stem cells are the primary tirgct in basic gene therapy research. The goal is to insert a functional gene into a self-renewing cell which, in turn, can divide and reconstitute the entire hemaiopoieUc system. In our in vivo model using beigc/nudc/xid (bnx) mice as transplant recipients and the technique of inverse PCR to identify the number of marked cells, we demonstrated the rare transaction of a human stem cell (Nolta et al., PNAS 93 in press, 1996). We have now assessed the number of marked progenitor or stem cells able to generate clonogcmc progeny capable of long-ierm expression of the inserted gene. CD34 progenitors were isolated from human bone marrow and transduced with retro v irai supernatant (LN) and stromal support as described (Nolta et al., Blood 86 101-110. 1995). The cells were co-transplanted with stroma producing human 1L-3 into bnx mice and allowed to engraft for 7-11 months, then the mice were sacrificed and bone marrow cells harvested. The cells were plated in human-specific CPU assay W- the selective agent G418. Two weeks later, colonies grown in G418 were plucked and PCR for the neo gene was done. We performed inverse PCR on neo colonies to analyze the integration site of the LN provirus in the human DNA. Genomic DNA isolated from each colony was digested with Taq 1, which cut once in the vector internal to each LTR and randomly cut in the human DNA flanking the vector. Two fragments of unique length were obtained per provirat integrant, each containing one intact LTR and a human DNA segment of variable size. The cut DNA segments were self-ligated and subjected to nested PCR, initially amplifying outward from ihe ends of the LTR into the flanking cellular DNA. Bands with similar electrophoretic mobility were isolated and scquenced to confirm identity. We assessed the ctonal diversity of G41g human CPU recovered from 18 long-term engrafted bnx mice, and found two to six human hematopoietic precursors to be generating all marked colony-forming progenitors (9 - 28) recovered from each animal. We searched GenBank for the human DNA sequences into which the vectors had integrated. In most cases, integration had occurred in unidentified human DNA. In one instance, we found a provirus integrated into the gene for human natriuretic factor. From 22 sets of experiments, we found pro viral integrants at identical sites in only two mice transplanted with cells transduced in [he same flask, indicating that a long-lived, marked cell had self-renewed in vitro prior to transplantation. Our in vivo model and the inverse PCR technique thus have allowed us to trace and identify the number of marked human cells capable of long-term generation of clonogenic progeny.

Original languageEnglish (US)
Pages (from-to)1057
Number of pages1
JournalExperimental Hematology
Volume24
Issue number9
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

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