Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability

 Insulin-like growth factor (IGF)-I and vanadate increase Na-dependent phosphate (Na/P_i) cotransport in opossum kidney (OK) cells. To gain more information about the mechanisms by which IGF-I and vanadate stimulate Na/P_i-cotransport, we measured type II Na/P_i-cotransporter (NaPi-4) protein abundance by Western blot analysis and investigated the effects of protein synthesis and tyrosine kinase inhibitors. The key findings in the present studies are as follows. First, incubation in IGF-I (10^–8 M) and/or vanadate (10^–3 M) for 3 h led to a non-additive 1.4-fold increase in Na/P_i-cotransport activity which was paralleled by a 1.5- to 2-fold increase in NaPi-4 protein. Second, actinomycin D did not abolish the increase in Na/P_i-cotransport and cycloheximide did not prevent the IGF-I-induced increase in Na/P_i-cotransport and NaPi-4 protein. Third, among the protein kinase inhibitors tested, only staurosporine substantially reduced the stimulation of Na/P_i-cotransport. In conclusion, the stimulatory effect of IGF-I on Na/P_i-cotransport is paralleled by an increased expression of NaPi-4 protein that is independent of protein synthesis and therefore results from increased protein stability. The observation that IGF-I and/or vanadate lead to similar increases in Na/P_i-cotransport and NaPi-4 protein abundance provides further evidence that the stimulation of Na/P_i-cotransport by IGF-I and vanadate involves protein tyrosine phosphorylation of the same signalling molecules. Received: 1 May 1998 / Received after revision: 25 August 1998 / Accepted: 1 September 1998     

Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Association between HLA and Japanese patients with rheumatoid arthritis

Japanese patients with rheumatoid arthritis (RA) were observed to have a statistical association with HLA-DR4, MT3. Strong association between the clinical severity of RA and HLA was also observed. Male patients had a stronger association with HLA than female patients. Males are more resistant to RA than females. This suggested that the threshold of liability for RA is higher in males than in females. Japanese patients with RA with systemic vasculitis were negative for HLA-Bw44 and had antilymphocytotoxic autoantibody, indicating that RA with systemic vasculitis is different in etiology from RA without systemic vasculitis.     

Ontogeny of J chain expression in pig lymphocytes

The ontogeny of lymphocytes expressing J chain in the cytoplasm (J+) was studied in pig foetuses by the immunofluorescent technique. Peripheral blood lymphocytes were the first J+ cells in prenatal life. The spleen and lymph nodes contained J+ cells in the last days of gestation. J+ cells were found in the lamina propria of the gut and some glands of conventional but not of germ-free piglets. J chain was not detected on or in cell membranes at any developmental stage.     

β Chain deficiency in three patients with dysfunctional C8 molecules

Structural and functional studies were performed on a dysfunctional C8 molecule present in the serum of two siblings and an unrelated individual. The C8 in these three sera exhibited a pattern of partial immunologic identity with C8 in normal serum but was devoid of functional activity. The C8 was immunoprecipitated from the three sera and from a control serum with an antihuman C8 antiserum and analyzed by SDS-PAGE using highly purified human C8 as a reference. A selective absence of a band of 62,000 mol. wt was observed in the immunoprecipitates from the sera containing dysfunctional C8. Experiments performed with the purified α-γ and γ subunits showed that the hemolytic activity of the C8 deficient sera could be reconstituted by the addition of the β chain but not the α-γ dimer. Binding of the dysfunctional C8 to C$\text{567}$ was excluded by the following observations: (1) EAC 1–7 treated with the C8 deficient sera and then washed could not be lysed after the addition of the β subunit and C9; and (2) the abnormal molecules did not interfere with the consumption of normal C8 by the soluble complex SC5b-7.     

31P and23Na nuclear magnetic resonance study on forebrain ischemia in rats with shift reagent Dy(TTHA)

³¹P and ²³Na nuclear magnetic resonance (NMR) spectroscopy was employed to study the dynamic changes in intracellular high-energy phosphates and sodium during 15min of forebrain ischemia and recirculation in in vivo rat brain. In the presence of the shift reagent Dysprosium triethylenetetramine-N,N,N,N,N,N-hexaacetic and [Dy(TTHA)], the sodium peak separated into two peaks, unshifted and shifted. During 15min of ischemia, the unshifted sodium peak decreased and the shifted sodium peak increased. With recirculation, the unshifted and the shifted sodium peaks returned to the preischemia level within 10min, but the shifted one increased during 30–60min. Intracellular high-energy phosphates and intracellular pH (pHi) decreased during 15min of ischemia and returned to the preischemia levels within 20min of recirculation. We conclude that the decrease in unshifted sodium peak during ischemia is due to the decrease in subarachnoid sodium and the cellular influx of interstitial sodium would be minimum. The increase in shifted sodium peak during ischemia is considered to be due to the dilatation of cerebral blood vessels and the increase in interstitial sodium which was transported from subarachnoid space.(Kurata M: ³¹P and ²³Na nuclear magnetic resonance study on forebrain ischemia in rats with shift reagent Dy(TTHA). J Anesth 7: 325–333, 1993)