Genetic construction by chromosomal gene transfer of mouse cell clones which harbor a transferred selected gene firmly associated with a nonhomologous chromosome

1978 ◽  
Vol 22 (1-6) ◽  
pp. 602-605
Author(s):  
K. Willecke ◽  
R. Mierau ◽  
A. Krüger ◽  
R. Lange
Keyword(s):  
Gene Transfer ◽  
Mouse Cell ◽  
Chromosomal Gene ◽  
Cell Clones ◽  
Nonhomologous Chromosome

Assay of mouse-cell clones for retrovirus p30 protein by use of an automated solid-state radioimmunoassay

Virology ◽  
1979 ◽  
Vol 97 (2) ◽  
pp. 464-467
Author(s):  
Stephen J. Kennel ◽  
Raymond W. Tennant
Keyword(s):  
Solid State ◽  
Mouse Cell ◽  
Cell Clones

322 POTENTIAL OF PRIMARY PORCINE KIDNEY CELLS FOR THE GENERATION OF TRANSGENIC PIGS VIA SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 308
Author(s):  
A. Wuensch ◽  
A. Richter ◽  
M. Kurome ◽  
B. Kessler ◽  
V. Zakhartchenko ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Homologous Recombination ◽  
Somatic Cell ◽  
Single Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Porcine Kidney ◽  
Cell Clones ◽  
Donor Cells ◽  
Additive Gene

The generation of genetically tailored pig models for biomedical research using somatic cell nuclear transfer (SCNT) is an efficient and precise approach, whereas the outcome is crucially dependent on the source of nuclear donor cells. Especially for site-directed mutagenesis by homologous recombination, including the generation of single cell clones, the demands on the target cells are high. Different primary cells used for SCNT have been tested for their efficiency in SCNT experiments, but further characterisation of the specific cell types, their morphology, proliferation, lifespan, and stability of karyotype is mostly lacking. We have evaluated the potential of 2 primary porcine kidney cell lines (PKC) isolated from juvenile pigs by a simple collagenase digestion and culture in collagen-coated dishes as cell source for SCNT, including their morphology, proliferation capacity, transfection efficiency, and capacity to support full-term development of SCNT embryos after additive gene transfer or homologous recombination. Single cell clones generated by subcloning of PKC at passage 3 showed different morphologies, proliferation rates, and lifespan, indicating that PKC culture is a mixed population of different types of fibroblasts and/or other cells types. The PKC could be maintained in culture for up to 71 passages without signs of senescence and decreased proliferation, exhibiting a stable karyotype containing 74% normal chromosome numbers (2N = 38) determined from metaphase spreads. In contrast, porcine fetal fibroblasts (PFF) and porcine ear fibroblasts (PEF) could be not be passaged more than 20 times. The calculation of growth curves at passage 4 to 5 showed that PKC exhibited a higher proliferation rate with a population doubling time of 16.6 to 18.4 h compared with PFF (23.2. h) and PEF (32.9 h). Furthermore the determination of the developmental competence after SCNT using PKC at passage 4 in 3 independent experiments and in vitro cultivation for 7 days resulted in a higher blastocyst rate (21%) compared with that in PFF (9.1%) and PEF (4.3%). The comparison of different transfection methods (lipofection, nanofection, conventional electroporation, nucleofection), using an expression vector for green fluorescent protein (GFP), showed that the NucleofectorTM technology gave the best results with transfection efficiencies of 70 to 89%, high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of stress. So far, around 150 cloned piglets using 18 different gene constructs have been produced using stable transfected PKC after additive gene transfer and targeting of 3 different loci. These findings demonstrate that among the 3 tested types of donor cells, PKC, PFF, PEF, primary PKC have outstanding potential for the production of genetically modified pigs by SCNT. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung, and Mukoviszidose e.V.

Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5539-5539
Author(s):  
Xianzheng Zhou ◽  
Xin Huang ◽  
Andrew C. Wilber ◽  
Lei Bao ◽  
Dong Tuong ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Cell Clones ◽  
Stable Gene Expression

Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p<0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p<0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

scholarly journals Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients.

1988 ◽  
Vol 167 (4) ◽  
pp. 1442-1458 ◽  
Author(s):  
D Wilkinson ◽  
R R de Vries ◽  
J A Madrigal ◽  
C B Lock ◽  
J P Morgenstern ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Class Ii ◽  
Cdna Clones ◽  
Mycobacterial Antigen ◽  
Differential Analysis ◽  
T Cell Clones ◽  
L Cells ◽  
Hla Dr ◽  
Cell Clones

We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation.

Generating of Human CD4+ and CD8+ T Lymphocytes toward NY-ESO-1 by T Cell Receptor (TCR) Modification and Transduction for Adoptive TCR Gene-Transfer

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3533-3533
Author(s):  
Holger Krönig ◽  
Kathrin Hofer ◽  
Daniel Sommermeyer ◽  
Christian Peschel ◽  
Wolfgang Uckert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
T Cell Receptor ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
Cell Clones ◽  
T Cell Clone ◽  
T Cell Transfer

Abstract The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.

Recombination of Endogenous TCR Chains with Retrovirally Introduced TCR Chains Can Result in Mixed T Cell Receptor Dimers Harbouring Harmful Alloreactivity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 823-823
Author(s):  
Marleen M Van Loenen ◽  
Renate de Boer ◽  
Gerdien L Volbeda ◽  
Avital L Amir ◽  
Renate S. Hagedoorn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Beta Chain ◽  
Antigen Specificity ◽  
Alpha Chain ◽  
Cell Clones ◽  
Engineered T Cells

Abstract T cell receptor transfer to engineer tumor specific T cells is being explored as a strategy for adoptive immunotherapy. By retroviral introduction of T cell receptors (TCRs), large numbers of T cells with defined antigen specificity can be obtained. The in vivo efficacy of adoptively transferred TCR engineered T cells has been demonstrated in mouse studies and recently the first clinical trial with TCR engineered T cells was performed in melanoma patients. However, a potential drawback of TCR gene transfer is the formation of mixed TCR dimers. Chains of the introduced TCR can pair with the endogenous TCR chains, resulting in unknown specificities, and potentially in harmful reactivity against patient HLA molecules. We investigated whether TCR gene transfer leads to the generation of new detrimental reactivities by creating T cells that expressed mixed TCR dimers. To be able to discriminate between the antigen specificity of the mixed TCR dimers and the introduced as well as the endogenous TCR, we transduced mono-specific T cells with seven different antigen specific TCRs. As mono-specific T cells we used CMV-pp50 specific HLA-A1 restricted T cells. The transduced T cells were analyzed for newly acquired specificities against a large HLA-typed EBV-LCL panel covering almost all HLA class I and II molecules. We transduced several polyclonal virus specific T cell populations with the seven different antigen specific TCRs, and showed that in all T cell populations at least one of the seven TCR-transduced populations acquired new alloreactivities. Furthermore, by randomly combining TCR alpha and beta chains derived from different T cell clones we created 60 mixed TCR dimers of which 17 acquired alloreactivity. These results indicate that recombination of the introduced TCR chains with the endogenous TCR chains frequently gives rise to new allospecificities. To ascertain that the newly acquired alloreactivities were exerted by mixed TCR dimers, we introduced only TCR alpha or beta chains into CMV-pp50 specific monoclonal T cells, and demonstrated for example, that the introduction of a CMV pp65 specific TCR alpha chain led to a newly acquired reactivity that was HLA B58 restricted. The introduction of only the beta chain of a minor histocompatibility antigen (mHag) HA-1 specific TCR led to a newly acquired HLA B52 specific reactivity. Furthermore, we analyzed whether mixed TCR dimers consisting of conserved TCRs with the same specificity could acquire new harmful reactivity. We recombined mHag HA-2 specific TCR alpha and beta chains from 4 different T cell clones. Of the 12 mixed TCR dimers, a combination of the mHag HA-2 specific TCR alpha chain derived from the HA2.6 T cell clone with the mHag HA-2 specific beta chain of clone HA2.19 resulted in alloreactivity that was HLA DQ3 restricted. These results indicate that each recombination of TCR chains after TCR gene transfer can potentially result in a harmful new reactivity. In conclusion, mixed TCR dimers due to pairing of endogenous TCR chains with introduced TCR chains acquire potentially dangerous reactivities, both class I and class II restricted. To limit the chance of generating self- or alloreactive T cells, TCRs may be constructed allowing selective pairing of the TCR alpha chain with the corresponding TCR beta chain. Alternatively, we propose to use virus specific T cells as host cells for TCR gene transfer. Since they consist of a restricted TCR repertoire, the number of different chimeric TCRs formed will be limited. By introducing into these T cells as controls only the alpha or beta chain of the TCR of interest, the reactivity of these T cells and harmful reactivities of the mixed TCR dimers can be tested against different patient derived cell types.

Chromosomal gene transfer: prospects of a new method for mapping closely linked human genes

1976 ◽  
Vol 16 (1-5) ◽  
pp. 405-408 ◽  
Author(s):  
K. Willecke ◽  
P.J. Davies ◽  
T. Reber
Keyword(s):  
Gene Transfer ◽  
New Method ◽  
Chromosomal Gene ◽  
Human Genes
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