Abstract
Obtaining sustainable source of cells or DNA from a single individual has always been a constraint in biomedical research. Generation of lymphoblastoid cell lines (LCLs) as a surrogate for replacement of isolated peripheral blood mononuclear cells (PBMCs) has substantially accelerated the process of biological investigations. LCLs can be established by transfection of B lymphocytes from peripheral blood with Epstein Barr Virus (EBV), bearing minor genetic and phenotypic alterations. Here we report the generation of LCLs for the indigenous population (Orang Asli) from Peninsular Malaysia. The establishment of cell line is crucial for such isolated populations since the population size is small and sample collections are often challenging. We applied an alternative approach in isolation of PBMCs before proceeding to EBV transfection of Orang Asli samples, of which the BD Vacutainer CPT tubes were used to replace the conventional protocol such as Ficoll-paque or histopaque. We called this approach the “modified method of CPT” (MMC). A total of 59 out of the 64 samples (92%) of Orang Asli were successfully transformed into LCLs by transfecting with EBV supernatant in a single attempt. To verify the success of LCLs transformation and confirm the presence of the human DNA, FCGR3B gene copy number variations of the DNA extracted from the LCLs were typed using Paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR) analysis. The genotype results were compared between the DNA obtained from the LCLs and the original PBMCs from the identical individuals, and results revealed 100% concordance rate. The success of the transformation for LCL for the indigenous populations will eventually allow a more comprehensive and advanced biomedical research to be carried out in the next future without the exhaustion of the biomaterials.
Highlights
Orang Asli is a generic Malaysian term used officially to name the indigenous people of Peninsular Malaysia
We report in this paper the generation of the immortalized lymphoblastoid cell lines (LCLs) for the Orang Asli population from Peninsular Malaysia using the modified method called modified method of cell preparation tube (CPT)” (MMC) and subsequently verified the integrity of genomic DNA extracted from the immortalized LCL samples by typing the FCGR3B copy number variation
Peripheral blood samples were collected in ACD tubes and kept for 3 days at the sampling locations without proper storage equipment
Summary
Orang Asli represents only 0.5% of the total Malaysian population and is classified into three major tribes, namely Negrito, Senoi and Proto Malay [3]. Some tribes for examples the Negritos, have been experiencing isolation and inbreeding resulted a smaller gene pool and population size [2,4]. They are thought to be an ideal model for the population genetic studies and mapping for complex diseases due to the fact that they are less admixed, less exposed to massive environmental changes and long period of isolation [5,6]. Establishing the immortalized lymphoblastoid cell lines (LCLs) is of crucial to provide infinite resources of Orang Asli population such as Negritos for future sustainable genomic studies
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
