Site‐directed mutagenesis of the human D antigen: definition of D epitopes on the sixth external domain of the D protein expressed on K562 cells

Abstract

The data provide strong evidence for the critical involvement of three amino acids, Asp350, Gly353, and Ala354, in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. This domain also appears to be essential for the expression of epD1, epD2, and epD4, as a loss of expression of these epitopes was observed in K562 cells transduced with the Dmut construct (encoding His350, Trp353, and Asn354). The K562/Dmut cell line has an identical molecular and serologic profile as the red cell D(IVb) phenotype, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by which to further map epitopes of D.

The Single Antigen expressing Lines (SALs) Concept: An Excellent Tool for Screening for HLA-Specific Antibodies

“…The concept of producing tailor-made target cells for serologic, cellular or biochemical analyses had been shown to be useful, e.g., Rh serology [20]. Employing the SALs developed according to this idea for HLA serology will have considerable impact for patients on transplant waiting lists, because of the possibility to define both the unacceptable as well as the AMs for patients with complex antibody reactivities.…”

Silencing and overexpression of human blood group antigens in transfusion: Paving the way for the next steps

“…Despite the hypothetical value of a model close to the natural context with expression of antigens on RBCs, most genetic manipulation related to transfusion over the last 30 years has been carried out on more or less heterologous systems and has yielded data that has proven relevant in transfusion practices. Many studies have used human hematopoietic cell lines such as the K562 cell line derived from an erythroleukemia, the HEL myeloid leukemia cell line, KU812E derived from Chronic Myeloid Leukemia, or MEG-01 cells derived from megacaryoblastic leukemia [5][6][7][8][9][10][11]. Indeed while it would in theory provide a favorable molecular context for expression of the studied antigen [8,12], the use of human cells close to the erythroid lineage can be difficult due to native expression of the studied proteins by the cell line [13], and genetic manipulation of hematopoietic cell lines is often labor intensive when plasmid transfection techniques are used.…”

“…While plasmid transfection remains the most widely used technique, some studies using delivery tools derived from Adeno-Associated Virus [30], from Human Immunodeficiency Virus type-1 [7,[31][32][33], or from Murine oncoretroviruses [5,6] have been reported. Viral vector techniques are based on the use of a defective viral genome to deliver genetic material such as a RNA or DNA expression unit as a non integrated element in the target cell when using AAV derived vectors and as integrated in the genome of the target cell with retrovirus derived vectors [34][35][36].…”

“…Viral vector techniques are based on the use of a defective viral genome to deliver genetic material such as a RNA or DNA expression unit as a non integrated element in the target cell when using AAV derived vectors and as integrated in the genome of the target cell with retrovirus derived vectors [34][35][36]. A notable advantage of retroviral vectors is to facilitate genetic manipulation procedures with high gene transfer efficiency and strong transgene expression in cell lines [5][6][7] and primary erythroid cell cultures [31,32] as compared to conventional transfection techniques. Microinjection of RNA transcribed in vitro, a seldom-used technique has been described for study of the JK (a.k.a.…”

“…Stimulation of beta-D-galactoside 2-alpha-L-fucosyltransferase expression in transfected A431 and COS1 cells allowed characterization of the locus for the H blood-group system [18]. Overexpression of RHD and RHCE gene products in K562 cells after genetic manipulation using murine retroviral vectors identified the molecular basis of the RH group [5,6,8,38]. Mapping of epitopes associated with the CROM (a.k.a.…”