Title Page
요약
Abstract
Contents
1. Introduction 15
1.1. Therapeutic monoclonal antibody 15
1.1.1. Monoclonal antibody 15
1.1.2. Therapeutic monoclonal antibody 15
1.2. Recombinant cell lines 16
1.2.1. Chinese hamster ovary (CHO) cells 16
1.2.2. Cell line development 17
1.3. Short interspersed nuclear elements (SINEs) 18
2. Objective 20
3. Material and Methods 24
3.1. Genomic DNA extraction of recombinant CHO cells 24
3.2. Alu PCR 25
3.3. DNA cloning and sequence analysis 26
4. Results and Discussions 28
4.1. Experimental design construction of Alu PCR 28
4.2. Transgene integration site identification 35
5. Conclusion 45
6. References 46
Table 1. SINEs used in Alu PCR 19
Table 2. Primers designed for Alu PCR 23
Table 3. Valid sequencing data of Alu PCR between clone 49c and cox4 33
Table 4. Whole sequencing data of Alu PCR between G-repeat and CTLA4Ig 38
Table 5. Whole sequencing data of Alu PCR between clone 49c and GSR 43
Figure 1. Overview of Alu PCR. (A) The loci between transgene and SINEs are amplified in first PCR step. Uracil-containing customized tail (dotted line) is attached to the primers which are complementary with SINEs. 100 pmol of transgene-complementary primer and 10... 22
Figure 2. The structure of primer including uracil. Customized tail (dotted line) is not complementary with genomic DNA. 30
Figure 3. Uracil detaching processes by UDG during uracil digestion step. (A) The initial product after first PCR step. (B) Structure of intermediate product after UDG treatment. Uracils are removed by UDG and apurinic sites are formed. (C) Final state of... 31
Figure 4. Alu PCR applied to endogenous gene. (A) The structure of cox4 (gray box) and clone 49c. Size between both primers on second coding... 32
Figure 5. Sequencing signals of Alu PCR result between cox4 and clone 49c. Black arrows mean start point and final point of reliable result. The locus between 331 and 992 is not shown. 34
Figure 6. The structure of two hypotheses. (A) The first case that SINE exists in front of transgene. (B) The second case that SINE exists behind transgene. 36
Figure 7. Alu PCR results in case that SINE exists in front of transgene. (A) Alu PCR results using CMV promoter on genome of two cell lines (Alb-EPO... 37
Figure 8. Sequencing signals of Alu PCR result between G-repeat and CTLA4Ig. Black arrows mean start point and final point of reliable result. The locus between 329 and 994 is not shown. 39
Figure 9. Alu PCR results in case that SINE exists behind transgene. (A) Alu PCR results using bGH poly A tail of GSR cell line. One remarkable result... 42
Figure 10. Sequencing signals of Alu PCR result between clone 49c and GSR. Black arrows mean start point and final point of reliable result. The locus between 341 and 672 is not shown. 44