Recent developments in genomics and proteomics have necessitated a constant supply of DNA belonging to specific genotypes. This requirement has taken an unusual dimension due to the relevance of genomics to medicine. Immortalization by EBV is an effective procedure for inducing long-term growth of human B-lymphocytes and these EBV-transformed cells can serve as the most reliable source of DNA. This service has added value if you are engaged in any clinical trials.
Transformation of peripheral blood lymphocytes by EBV is a laboratory procedure developed as a combination of a sound scientific operation and state of the art technology involving tissue culture. Immortalization of cells takes 6-8 weeks to generate large amounts of cells that can be indefinitely grown as a continuing source of genetic material. We shall be delighted to serve your needs if you are engaged in any research involving DNA. You simply need to supply us with a sample of whole blood.
EBV TRANSFORMATION
A generalized protocol for EBV Transformation of peripheral blood lymphocytes includes following steps.
1. The peripheral blood lymphocytes obtained from a donor are cultured in tissue culture medium RPMI 1640 supplemented with 10% fetal bovine serum and essential amino acids.
2. The culture is infected with EBV supernatant. The cells usually start showing morphological changes after 3 to 4 days when dividing cells can be seen as dumbbell shaped structures under an inverted microscope.
3. Typical morphological changes manifested by an actively growing cell culture comprise cellular clumps which can be seen with a naked eye. Usually it takes six to eight weeks to obtain a fully transformed culture showing typical manifestation of big cellular masses (Figures 1-4).
What KTI will need from you
- Sample and Shipping Requirement: The whole blood sample (12-15 ml) collected in a sterile citrate or heparin tube. The blood sample must be stored at room temperature and shipped to KamTek’s Beechcraft Facility within 24 hours.
- Donor Consent Form: It is essential to have properly executed donor consent form approved by the institutional review committee if the DNA is going to be used for research.
What KTI can provide you
- We will establish the lymphoblast B-cell lines for you.
- We will provide you with the data on viability of normal as well as transformed cells.
- We will also provide you with the DNA profiles of the normal as well as transformed cells if you need.
- KTI maintains a state of the art repository where we can store your samples as long as you desire for a nominal fee.
- Cryo-preservation is done with the use of Cryo-med which assures controlled temperature freezing and consequently higher viability of the cryo-preserved samples (Table 1).
| Table 1. Viability of Normal and Transformed Cells | ||
| Sample No. | Normal Cells(Viability %) | Transformed Cells(Viability %) |
| 1 | 93 | 88 |
| 2 | 100 | 88 |
| 3 | 89 | 90 |
| 4 | 73 | 90 |
| 5 | 86 | 94 |
| 6 | 87 | 94 |
| 7 | 89 | 97 |
| 8 | 92 | 94 |
