Publication: Kan hücrelerinin izolasyonu ve transfeksiyonu
Abstract
İmmunoporasyon, farklı tip hücrelerin immunolojik özelliklerinden yararlanılarak yapılan yeni bir transfeksiyon metodudur. Antikor kaplı manyetik boncuklarla farklı tip hücrelerin, uygun transfeksiyon ortamında karışması sonucu, minimum hücre ölümü ve yüksek transfeksiyon etkinliği gözlenmiştir. İmmunoporasyonla şimdiye kadar sadece doku kültürü hücreleri transfekte edilmiştir. Bu çalışmanın amacı, immunoporasyon metoduyla kandaki mononükleer hücrelere DNA transferini sağlamaktır. Mononükleer hücreler, seyreltilmiş periferik kandan Nycoprep (1.077 g/ ml) kullanılarak izole edildi ve fitohemaglutinin ile 24 saat aktive edildi. Aktive edilmiş mononükleer hücreler, Anti-Class I kaplı manyetik boncuklar ve plazmid DNA (pEGFP-N1) ile oda sıcaklığında bir gece karıştırıldı. Sonra boncuğa bağlı olmayan hücreler bir manyetik separatörle ayrılarak %10 fetal sığır serumu içeren RPMI-1640 besi ortamı içinde 37 oC'de 48 saat inkübe edildi. Daha sonra hücreler PBS'le (fosfatla tamponlanmış salin) yıkanarak poli-L-lizin kaplı kuyucuklu lamlara transfer edildi. Fluoresan mikroskop altında incelenen örneklerde yeşil fluoresansın varlığı gözlendi. Görülen fluoresans, DNA'nın mononükleer hücrelere girip ekspresyon yaptığının kanıtıdır. Sonuç olarak, bu çalışma immunoporasyon tekniğinin mononükleer hücrelere DNA transferi için kullanılabileceğini göstermektedir.
ISOLATION OF BLOOD CELLS AND TRANSFECTION Immunoporation is a novel transfection method based on their immunological identity of different types of cells. Mixing different types of cells with antibody coated magnetic beads results high level of transfection with minimal cell death in the presence of a desired transfection agent. So far only tissue culture cells have been transfected by immunoporation. The purpose of this study is to transfect mononuclear cells (MNC) with DNA. MNCs were isolated from diluted human peripheral blood using Nycoprep (1.077 g/ ml) density gradient media and then they were activated by phytohemagglutinin for 24 hrs. At room temperature the activated MNCs were mixed overnight with Anti-Class I coated magnetic beads in the presence of plasmid DNA (pEGFP-N1). After the separation of bead-free cells from others by a magnetic separator, the bead-free cells were incubated in culture medium (RPMI-1640 containing 10% fetal calf serum) for 48 hrs at 37 oC. The MNCs were washed with phosphate buffered saline (PBS), transferred to poly-L-lysine coated slides. Gene expression was detected using fluorescent microscopy. Observed fluorescence is an evidence of entrance of DNA into the cells. In conclusion in this study the detection of the gene expression suggests that immunoporation technique may be used for transfection of peripheral blood cells.
ISOLATION OF BLOOD CELLS AND TRANSFECTION Immunoporation is a novel transfection method based on their immunological identity of different types of cells. Mixing different types of cells with antibody coated magnetic beads results high level of transfection with minimal cell death in the presence of a desired transfection agent. So far only tissue culture cells have been transfected by immunoporation. The purpose of this study is to transfect mononuclear cells (MNC) with DNA. MNCs were isolated from diluted human peripheral blood using Nycoprep (1.077 g/ ml) density gradient media and then they were activated by phytohemagglutinin for 24 hrs. At room temperature the activated MNCs were mixed overnight with Anti-Class I coated magnetic beads in the presence of plasmid DNA (pEGFP-N1). After the separation of bead-free cells from others by a magnetic separator, the bead-free cells were incubated in culture medium (RPMI-1640 containing 10% fetal calf serum) for 48 hrs at 37 oC. The MNCs were washed with phosphate buffered saline (PBS), transferred to poly-L-lysine coated slides. Gene expression was detected using fluorescent microscopy. Observed fluorescence is an evidence of entrance of DNA into the cells. In conclusion in this study the detection of the gene expression suggests that immunoporation technique may be used for transfection of peripheral blood cells.