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[Expression of human multidrug resistance gene (mdr1) cDNA in murine ES cells and in chimeric mice]
Abstract:
Human multidrug resistance gene (mdr1) was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in colchicine concentration (30, 50, 100, and 200 ng/ml respectively). Finally, we obtained 4 clones that could be stably grown in culture medium with colchicine at 200 ng/ml and designated as ES-mdr1 clones A, B, C, and D. Southern blot analysis of DNA from ES-mdr1 A and D cells digested by Hind III and hybridized with mdr1 cDNA 5 A probe was shown in Fig. 3. Characteristic 4.8 and 2.4 kb fragments of mdr1 gene were found as expected and their amplification under increased concentration of colchicine in culture medium was also evident from the figure. Slot blot and Northern analysis of total RNA and poly A+ RNA extracted from ES-mdr1 cells were shown in Fig. 4 and 5, demonstrating that ES-mdr1 cells could express mdr1 mRNA. Indirect immunofluorescence analysis with antibodies against p170 glycoprotein indicated that p170 protein translated from mdr1 mRNA was present at the surface of ES-mdr1 cells (Plate I, Fig. 2). The biological characteristics of ES-mdr1 cells cultured in medium containing 200 ng/ml colchicine were investigated. The cells maintained their undifferentiated morphology and grew in nests (Plate I, Fig. 1), like the parental ES-5 cells. When ES-mdr1 cells were cultured in suspension in vitro, these cells were still capable of producing simple and cystic embryoid bodies. ES-mdr1 cells injected subcutaneously into 129 mice formed tumor-like outgrowths giving a great variety of cell types (Plate I, Fig. 4). These results indicated that the integration and expression of human mdr1 gene and selection against colchicine did not affect the pluripotency of ES-mdr1 cells both in vitro and in vivo. However, ES-mdr1 cells, unlike their parental ES-5 cells, could no longer be induced to differentiate by either RA or HMBA (Plate I, Figs. 3a, 3b), indicating that the human mdr1 gene transfected ES cells had changed their competence of inducible response to differentiation in vitro. The details and possible significance of such change require further studies. From the above preliminary data, we are of the opinion that ES-mdr1 cells may serve as a model to study the mode of action of p170 glycoprotein at cellular level and to screen possible means to counteract the action of mdr1 gene.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Transfected mouse embryonic stem cells with the human multidrug resistance gene (mdr1) maintained pluripotency but lost inducible differentiation capacity. These ES-mdr1 cells offer a model for studying mdr1 gene action and potential countermeasures.
Area of Science:
- Molecular Biology
- Developmental Biology
- Stem Cell Biology
Background:
- The human multidrug resistance gene (mdr1) confers resistance to various chemotherapeutic agents.
- Understanding the impact of mdr1 gene expression on stem cell properties is crucial for gene therapy and cancer research.
Purpose of the Study:
- To investigate the integration and expression of the human mdr1 gene in mouse embryonic stem cells (ES-5 line).
- To evaluate the effect of mdr1 gene transfection on the pluripotency and differentiation potential of these cells.
- To establish a cellular model for studying the function of the mdr1 gene product, P-glycoprotein.
Main Methods:
- Calcium phosphate-mediated transfection of mouse ES-5 cells with the human mdr1 gene.
- Selection of transfected cells using stepwise increases in colchicine concentration.
- Confirmation of mdr1 gene integration and expression using Southern blot, slot blot, and Northern blot analyses.
- Assessment of P-glycoprotein expression via indirect immunofluorescence.
- Evaluation of pluripotency and differentiation capacity in vitro and in vivo.
Main Results:
- Successfully generated four stable ES-mdr1 clones expressing functional human mdr1 gene and P-glycoprotein.
- ES-mdr1 cells retained their undifferentiated morphology and pluripotency in vitro and in vivo, forming embryoid bodies and teratomas.
- Transfected cells showed resistance to colchicine.
- ES-mdr1 cells lost their ability to be induced to differentiate by retinoic acid (RA) or hexamethylene bisacetamide (HMBA).
Conclusions:
- The integration and expression of the human mdr1 gene in mouse embryonic stem cells do not compromise their pluripotency.
- Expression of the human mdr1 gene alters the differentiation competence of embryonic stem cells.
- ES-mdr1 cells serve as a valuable model for studying the cellular mechanisms of multidrug resistance and for screening potential therapeutic interventions.