B. Generation of LDHB conditional knockout mice
3. ES cell screening
a. Genomic DNA isolation by phenol-chloroform extraction
Transfection 된 ES cell(24well plate from 마크로젠)에 DNA lysis buffer( + proteinase K) 로 55℃에서 5-6 시간 동안 lysis 한다. Phenol-chloroform extraction 방법으로 ES cell 의 genomic DNA 를 추출한다.
b. Southern blot analysis
Phenol-chloroform extraction 을 통하여 얻어진 genomic DNA 를 BamH1(NEB)으로 overnight enzyme digestion 한다. 0.6% agarose gel 을 이용하여 electrophoresis 한 뒤 transfer 한다. transfer 가 끝난 membrane 은 2000J 로 cross-link(UV STRATALINKER® 1800)시킨다. LDHB N probe 를 사용하였으며 α-P32를 이용하여 labeling 하였다. -80℃에서 24 시간 동안 film exposing 하였다.
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4. Injection of ES cells into blastocystsES cell screening 을 통해 targeting vector 가 들어간 positive ES cell 을 선별하여
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6. Confirmation of LDHB knockout mice마지막으로 Cre mouse 와 mating 으로 얻어진 knockout mouse 들은 PCR genotyping 과 southern blot analysis 를 통해 확인한다. Genotyping 은 LDHB , Flp, 그리고 Cre 에 관하여 진행하였으며 southern blot analysis 는 BamHⅠ으로 digestion 하여 LDHB N probe 로 labeling 하였다.
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Ⅲ. 결 과
A. ES cell screening
Southern blot analysis 를 통해 targeting vector 가 들어간 positive ES cell 은 genomic DNA 를 BamHⅠ으로 digestion 하여 LDHB N probe 를 이용해 선별하였다. WT allele 은 12.2kb , targeting vector 는 7.5kb 에서 각각 확인 할 수 있었다(Fig. 4A).
Second confirmation 에서는 BamHⅠdigestion 수행하였으며 BamHⅠ은 LDHB N probe 를 이용하여 screening 하여 positive clone 을 얻었다(Fig. 4B).
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Fig. 4. ES cell screening by southern blotting (A)Analysis of control[C] and targeted[T]ES cells. Digestion of ES cell DNA with BamHⅠ generates a novel fragment of kb in LDHB+/neo-loxP clones. (B) Second confirmation of control and targeted ES cells.
Digestion of ES cell DNA with BamHⅠ(B). Southern blot analysis of control and targeted ES cells with LDHB N probe(B).
C T
- 12.2 kb - 7.5 kb
- 7.5 kb - 12.2 kb ES cell
A
B ES cell
C T T T T T T T
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LDHB+/neo-loxP mice(+/L) with chimera mice. Genotyping of WT mice by southern blot analysis (A) and PCR(B).- 21 -
C. Genotyping of LDHB+/neo-loxP, FLPGerm-line transmission 된 마우스와 Flp 를 지닌 마우스의 교배를 통해 태어난 마우스 중에서 Flp 를 포함하는 mouse (LDHB+/neo-loxP, FLP)를 LDHB primer 와 Flp primer 를 이용하여 PCR 을 통해 선별하였다. (Fig. 6A and 6B)
Fig. 6. Genotyping of LDHB+/neo-loxP, FLP mice (A) Analysis of wild-type(+/+) and LDHB+/neo-loxP mice(+/L) with LDHB primer. (B) Genotyping of LDHB+/neo-loxP, FLP mice with Flp primer
A
B
+/L +/L +/L +/L +/L +/L +/L +/L +/L +/L +/L +/L +/L
+/+ +/+
Flp
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
Flp Flp Flp Flp Flp Flp Flp Flp Flp
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D. Genotyping of LDHB+/loxP mice결과(C)에서 FLP 를 가지고 있는 마우스를 선별한 뒤 wild type 마우스와 mating 시켰다. FRT site 에 flanking 되어 있던 positive selection marker 인 neo-cassette 가 Fig. 7 과 같이 Flp 에 의해 제거 된 마우스를 얻었다.
Fig. 7. Genotyping of LDHB+/ loxP mice (A) Analysis of wild-type(+/+) and LDHB+/neo-loxP mice(+/L) with LDHB primer. (B) Genotyping of LDHB+/neo-LDHB+/neo-loxP, FLP mice with Flp primer.
A
B
+/L +/L
Flp Flp Flp
+/+ +/+ +/+ +/+ +/+
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E. Genotyping of LDHB knockout mice(LDHB+/loxP mice, Cre)
(D)와 마찬가지로 결과(C)에서 FLP 를 가지고 있는 마우스를 선별한 뒤 wild-type 마우스가 아닌 Cre-recombinase 를 가진 마우스를 mating 시킨다. 이 사이에서 태어난 마우스 중에서 Cre 를 가지고 있는 마우스를 선별하여 결과적으로 LDHB+/- 마우스를 얻었다.
Figure 8. Genotyping of LDHB+/ loxP, Cre mice (A) Analysis of wild-type(+/+) and LDHB+/neo-loxP mice(+/L) with LDHB primer. (B) Genotyping of LDHB+/neo-loxP, FLP mice with Flp primer. (C) Genotyping of Cre with Cre primer
A
B
C
+/L +/L +/L +/L +/L +/L
Cre Cre Cre Cre Cre Cre
Flp Flp Flp Flp
+/+ +/+ +/+ +/+
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Ⅴ. 결 론
본 연구에서는 마우스에서 LDHB 를 제거함으로써 세포내의 모든 lactate dehydrogenase 가 LDHA 로 구성되도록 즉, lactate dehydrogenase 의 활성이 항상 증가되도록 조작하고자 하였다.
본 연구에서는 마우스의 LDHB 게놈을 이용하여 LDHB 유전자 적중용 벡터를 제작하였으며, 이를 이용하여 LDHB 유전자를 마우스의 줄기배아세포 (embryonic stem cell)에서 적중하였다. 다음으로 LDHB 가 적중된 마우스 줄기배아세포를 대리모 마우스의 blastocyst 에 주입하여 chimera 마우스를 생산하였고, 이 chimera 마우스로부터 LDHB+/- 마우스를 얻었다.
현재 LDHB+/- 마우스가 태어나 homozygous 마우스를 만들기 위하여 교배 중이며, LDHB 가 적중된 마우스와 heart-specific 하게 Cre recombinase 가 발현되는 마우스를 교배하여 심장에서 LDHB 가 제거된 마우스를 제작하고 있다.
앞으로 LDHB knockout 마우스는 발암기전 연구를 위한 동물 모델 및 미토콘드리아 활성저하 동물 모델로 유용하게 사용될 것으로 기대된다.
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(Supervised by Assistant Professor Chan Bae Park)
In general, differentiated cells produce most of the ATP to growth from glucose through metabolism. Normal cells produce 2 ATP by glycolysis in the cytoplasm and convert to 2 pyruvates. Next, this pyruvate produces about 36 ATP by oxidative phosphorylation in the mitochondria.
On the other hand, many metastatic cancer cells have altered glycolytic(glucose) metabolism. The lactate released by this altered glycolytic metabolism exported out of cancer cells. Because the tumor environment is highly hypoxic so that cancer cells produce of ATP by glycolysis. Cancer cells uptake glucose much more than normal cells. Because, they use inefficient glycolysis for producing ATP. In cancer cell, expression of gene associated with glycolysis and enzyme about lactate production are increased for activating glycolysis.
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Recently, Many report presented about that discovered altered glucose metabolism in cancer cells is not passive alteration by change in environment, but important role in normal cells.
Lactate dehydrogenase(LDH) is a major enzyme involved in conversion of lactate in glycolysis. There are five LDH isozymes as a result of the five differenct combination that are produced by two subunit(A and B gene). Also, they have different enzymatic activity.
The LDH-1 that consist of 4 LDHA complex is most active in LDH activity. But LDH activity is inverse amounts of LDHB. So we want make the condition of increased LDHA activity without LDHB in the cell.
In this study, we designed a targeting vector about LDHB gene used by LDHB genome(mouse). And then, this targeting vector insert into the ES cell. Then targeted ES cell inject into blastocyst. As a result, we have got the chimera, LDHB targeted mouse. And now, we have gained the LDHB knockout mouse that generated by mating LDHB targeted mouse and Cre-expressed mouse.
We expect that LDHB knockout mouse is used to animal model of research for cancer research and mitochondrial function that involved in cancer cell metabolism.
Key words : Glycolysis , Oxidative phosphorylation , Lactate dehydrogenase , Conditional gene knockout mouse