Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative

1987 ◽  
Vol 7 (10) ◽  
pp. 3561-3565
Author(s):  
M M Seidman
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Restriction Analysis ◽  
Shuttle Vector ◽  
Recombination Reaction ◽  
Neomycin Resistance Gene ◽  
African Green Monkey Cells ◽  
The Individual ◽  
Neomycin Resistance

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

scholarly journals Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

1987 ◽  
Vol 7 (10) ◽  
pp. 3561-3565 ◽  
Author(s):  
M M Seidman
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Restriction Analysis ◽  
Shuttle Vector ◽  
Recombination Reaction ◽  
Neomycin Resistance Gene ◽  
African Green Monkey Cells ◽  
The Individual ◽  
Neomycin Resistance

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

Recombination events after transient infection and stable integration of DNA into mouse cells

1985 ◽  
Vol 5 (4) ◽  
pp. 659-666 ◽  
Author(s):  
S Subramani ◽  
J Rubnitz
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Transcription Unit ◽  
Sister Chromatid Exchanges ◽  
Neomycin Resistance Gene ◽  
Stable Integration ◽  
Mouse Cells ◽  
Chromatid Exchanges ◽  
Neomycin Resistance ◽  
Constructed Plasmids

To investigate the recombinational machinery of mammalian cells, we have constructed plasmids that can be used as substrates for homologous recombination. These plasmids contain two truncated nontandem, but overlapping, segments of the neomycin resistance gene, separated by the transcription unit for the xanthine guanine phosphoribosyl transferase gene. Recombination between the two nonfunctional neomycin gene sequences generates an intact neomycin resistance gene that is functional in both bacteria and mammalian cells. Using these plasmid substrates, we have characterized the frequencies and products of recombination events that occur in mouse 3T6 cells soon after transfection and also after stable integration of these DNAs. Among the chromosomal recombination events, we have characterized apparent deletion events that can be accounted for by intrachromatid recombination or unequal sister chromatid exchanges. Other recombination events like chromosomal inversions and possible gene conversion events in an amplification unit are also described.

scholarly journals Recombination events after transient infection and stable integration of DNA into mouse cells.

1985 ◽  
Vol 5 (4) ◽  
pp. 659-666 ◽  
Author(s):  
S Subramani ◽  
J Rubnitz
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Transcription Unit ◽  
Sister Chromatid Exchanges ◽  
Neomycin Resistance Gene ◽  
Stable Integration ◽  
Mouse Cells ◽  
Chromatid Exchanges ◽  
Neomycin Resistance ◽  
Constructed Plasmids

To investigate the recombinational machinery of mammalian cells, we have constructed plasmids that can be used as substrates for homologous recombination. These plasmids contain two truncated nontandem, but overlapping, segments of the neomycin resistance gene, separated by the transcription unit for the xanthine guanine phosphoribosyl transferase gene. Recombination between the two nonfunctional neomycin gene sequences generates an intact neomycin resistance gene that is functional in both bacteria and mammalian cells. Using these plasmid substrates, we have characterized the frequencies and products of recombination events that occur in mouse 3T6 cells soon after transfection and also after stable integration of these DNAs. Among the chromosomal recombination events, we have characterized apparent deletion events that can be accounted for by intrachromatid recombination or unequal sister chromatid exchanges. Other recombination events like chromosomal inversions and possible gene conversion events in an amplification unit are also described.

Regulation of pp60c-src synthesis by inducible RNA complementary to c-src mRNA in polyomavirus-transformed rat cells

1986 ◽  
Vol 6 (7) ◽  
pp. 2305-2316
Author(s):  
S Amini ◽  
V DeSeau ◽  
S Reddy ◽  
D Shalloway ◽  
J B Bolen
Keyword(s):  
Resistance Gene ◽  
Tumor Antigen ◽  
Kinase Activity ◽  
Src Kinase ◽  
Soft Agar ◽  
Transformed Cells ◽  
Neomycin Resistance Gene ◽  
Sense Orientation ◽  
Normal Rat ◽  
Neomycin Resistance

To determine the potential role of pp60c-src in polyomavirus-transformed cells, we constructed a recombinant plasmid with the mouse metallothionein-I promoter upstream of a src gene in an anti-sense orientation. We cotransfected this plasmid into middle tumor antigen-transformed FR3T3 cells with a plasmid containing the neomycin resistance gene, and G418 resistant colonies were selected. Analysis of these cells for pp60c-src expression revealed that 50 of the 200 cellular clones screened were found to have decreased levels of c-src expression when compared with the parental middle tumor antigen-transformed cells. Three independent clones which transcribed the expected 3.6-kilobase src complementary RNA and had levels of pp60c-src kinase activity comparable to that of normal FR3T3 cells were further analyzed. In the presence of Cd2+, these clones grew significantly slower in monolayer cultures than either the parental transformed cells (FR18-1) or FR18-1 cells transfected with the neomycin resistance gene alone. The morphology of these clones in the presence of Cd2+ was distinct from that of either the parental FR18-1 cells or normal FR3T3 cells. The clones expressing the complementary src RNA were found to form fewer colonies in soft agar, form fewer foci on monolayers of normal rat cells, and form tumors more slowly following injection into syngenic rats when compared with parental FR18-1 cells. The results of these studies suggest that the level of pp60c-src kinase activity affects the growth characteristics and transformation properties of polyoma virus-transformed rat cells.

Use of Allogeneic Bone Marrow Labeled with Neomycin Resistance Gene to Examine Bone Marrow-Derived Chimerism in Experimental Organ Transplantation

1997 ◽  
Vol 6 (4) ◽  
pp. 369-376 ◽  
Author(s):  
J. P. Smith ◽  
J. Kasten-Jolly ◽  
L. Rebellato ◽  
Carl E. Haisch ◽  
Judith M. Thomas
Keyword(s):  
Bone Marrow ◽  
Resistance Gene ◽  
Nonhuman Primate ◽  
Antithymocyte Globulin ◽  
Molecular Techniques ◽  
Allogeneic Bone ◽  
Nonhuman Primate Model ◽  
Primate Model ◽  
Neomycin Resistance Gene ◽  
Neomycin Resistance

Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neor) using the retroviral vector pHSG-Neo. Neor-Mransduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence of Neor-positive cells. Tissue samples obtained at necropsy also were assessed for Neor-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction using Neor sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.

scholarly journals Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3048-3057 ◽  
Author(s):  
CE Dunbar ◽  
M Cottler-Fox ◽  
JA O'Shaughnessy ◽  
S Doren ◽  
C Carter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Resistance Gene ◽  
Peripheral Blood ◽  
Marker Gene ◽  
Autologous Transplantation ◽  
High Dose ◽  
Neomycin Resistance Gene ◽  
Neomycin Resistance

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.

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