行政院國家科學委員會補助專題研究計畫 成果報告 期中進度報告. 基因剔除動物模式研究人類 L2DTL/ramp 基因

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1 行政院國家科學委員會補助專題研究計畫 成果報告 期中進度報告 基因剔除動物模式研究人類 L2DTL/ramp 基因 計畫類別 : 個別型計畫 整合型計畫計畫編號 :NSC B 執行期間 :91 年 5 月 1 日至 92 年 4 月 30 日 計畫主持人 : 許輝吉教授 共同主持人 : 林淑華教授 計畫參與人員 : 劉兆蓮潘弘偉賴寶蓮柳舒祥 成果報告類型 ( 依經費核定清單規定繳交 ): 精簡報告 完整報告 本成果報告包括以下應繳交之附件 : 赴國外出差或研習心得報告一份 赴大陸地區出差或研習心得報告一份 出席國際學術會議心得報告及發表之論文各一份 國際合作研究計畫國外研究報告書一份 處理方式 : 除產學合作研究計畫 提升產業技術及人才培育研究計畫 列管計畫及下列情形者外, 得立即公開查詢 涉及專利或其他智慧財產權, 一年 二年後可公開查詢 執行單位 : 台大醫學院病理研究所 中華民國 92 年 7 月 30 日

2 ( 二 ). 中英文摘要及關鍵詞 (keywords) Abstruct in Chinese : 關鍵詞 : 基因剔除 ; L2DTL; 果蠅 l(2)dtl; ramp; WD40 重複 ; 肝癌 人類 L2DTL 基因是因與果蠅 (Drosophila melanogaster) 的致命性基因 lethal (2) denticleless, 簡稱 l(2)dtl, 相似而命名. 果蠅之 l(2)dtl 基因是因果蠅若發生該基因之同型合子突變 (homozygous mutations), 其胚胎會缺失腹部齒狀帶 (ventral denticle belts), 且死亡 (lethal), 而得名 最近的唯一論文証實該基因蛋白確實是核蛋白, 與核基質相連, 表現於胚胎癌細胞 株 NT2, 但在 retinoic acid 誘發分化時, 則發生調降現象, 故稱為 ramp 蛋白. L2DTL/ramp 蛋 白是一個 WD-40 repeats 蛋白, WD-40 repeats 蛋白只見於所有真核細胞 (eukaryotes), 而 原核細胞 (prokaryotes) 則無, 且參與多種重要的生物學功能, 如細胞分裂, 細胞命運決擇 (cell-fate determination), 基因轉譯, 穿膜訊息傳遞 (transmembrane signaling), mrna 修 飾與小泡融合等. 雖然 L2DTL/ramp 蛋白在人類之功能仍然處於渾沌未明, 本研究室發現 L2DTL/ramp mrna 表現在大多數人類胚胎組織發育, 對胚胎組織發育上扮演非常重要角 色, 包括肝臟 而在 271 例單一原發性肝癌中, 我們發現 53% 的肝癌有 L2DTL/ramp mrna 的過度表現, 此種過度表現伴隨分級高 (P = ) 及分期高之腫瘤 (P = ), 十年之 存活率亦較差 (P = ) 我們的觀察顯示在 cytokinesis 與細胞增生伴有重要角色的 L2DTL/ramp, 也在人類胚胎組織發生上深具重要性, 酷似其果蠅 homologue 然則, 人類 以外的 homologue 尚未發現, 特別是 murine homologue, 因此,L2DTL/ramp 在胚胎組織 與胚胎發育上的真正生理功能仍然未明 L2DTL/ramp 在肝癌及一些常見癌症的過度表現 也顯示 L2DTL/ramp 在癌症的發生上可能的重要性, 也是一急待開發的領域 由此可見, L2DTL/ramp 的研究的重要性是多方面而影響可能是深遠的 以 homologous recombination 在小鼠 mice 胚胎幹細胞 (embryonic stem cell) 以轉殖基因 (transgene) 與基因剔除 (knockout) 技術產生的小鼠是研究發生學與腫瘤發生的理想模式 因 為基因之異位性表現 胚胎死亡 基因剔除而無表現型 (null mutations) 已可經反轉病毒載體 及組織特異性與條件性剔除 (conditional knockouts) 等修改而加以克服 由於轉殖基因與基因 剔除鼠在現代醫學的重要性, 及有鑑於 L2DTL/ramp 在胚胎發育與肝癌, 乃至其他人類癌 症之重要性, 本研究室在獲得本院唯一經驗豐富的林淑華教授之轉殖基因研究室之合作 下, 提出本計畫, 以轉殖鼠進行 L2DTL/ramp 基因之研究計畫 除了建立 L2DTL/ramp 轉 殖基因鼠之外, 也將以傳統基因轉殖技術破壞 L2DTL/ramp 基因, 並以 Cre/LoxP 系統做組 織特異性條件式剔除法破壞 L2DTL/ramp 基因, 建立轉殖 本研究之目標 : 以基因轉殖及基因剔除技術建立 L2DTL/ramp 基因過表現型及不表現型鼠 (heterozygous and homozygous knockouts), 以供各項研究之用 第一部份 : L2DTL/ramp 基因轉殖小鼠 (1) 以肝細胞專一性的前導子 (promoter) 建立人類 L2DTL/ramp 基因過表現型轉殖鼠 (2) 由於 L2DTL/ramp 基因在肝癌中所扮演的角色仍未知, 所以視實驗結果來分析轉殖小鼠 的表現型, 及以免疫組織化學染色法與形態法分析的研究以了解 L2DTL/ramp 基因在肝癌 上所扮演的角色 (3) 探討 L2DTL/ramp 基因在肝癌發生過程的角色 以肝癌致癌物 (hepatocarcinogens) 在 L2DTL/ramp 轉殖基因 (transgene) 鼠, 以組織病理方法探討 L2DTL/ramp 癌細胞轉移能力之 影響

3 第二部份 : L2DTL/ramp 基因剔除小鼠 (1) 觀察 L2DTL/ramp 在 homozygous deletion 時, 是否與果蠅一樣導致胚胎死亡, 以驗證二者是否是真正的 homologues 因人類 L2DTL 之 C' 端具有一段果蠅 l(2)dtl 或人類其他基因所無之特異序列, 可能發生不同的結果 (2) 探討 L2DTL/ramp 基因在胚胎發生過程中對各器官組織細胞生長 移動 分化的角色 以基因標的 (gene targeting) 及剔除法技術來建立不表現核 L2DTL/ramp 蛋白的轉殖鼠 為避免果蠅全身性基因剔除發生的胚胎死亡情形, 將以有條件與組織特異性的 L2DTL/ramp 基因剔除方式, 產生轉殖鼠

4 Abstruct in English : Keywords: Gene knockout; L2DTL; Drosophila l(2)dtl; ramp; WD-40 repeats; Hepatocellular carcinoma Human L2DTL is homologous to lethal (2) denticleless, l(2)dtl, initially identified in Drosophila melanogaster. Drosophila l(2)dtl is so designated because embryos with homozygous mutations of the gene lack ventral denticle belts and are lethal (12). L2DTL protein is a nuclear matrix-associated protein and is downregulated during RA-induced neuronal differentiation of NT2 cells, designated as ramp. L2DTL/ramp is a WD-40 repeats proteins that are found in all eukaryotes but not in prokaryotes, and involved in several important biologic functions. These include cell division, cell-fate determination, gene transcription, transmembrane signaling, mrna modification and vesicle fusion. We showed that L2DTL/ramp mrna plays important role in embryogenesis in most tissues, including liver. L2DTL/ramp mrna was overexpressed in 60% of 174 unifocal primary HCCs, and associated with high grade (P = ) and advanced HCC (P < ), and worse five-year survival (P = 0.025). Our observations indicate that L2DTL/ramp that plays important role in cell cytokinesis and cell proliferation also play important role in embryongenesis, resembling its Drosophilia homologue. Despite these observations, the murine homologue has not been identified, and the physiologic function of human L2DTL/ramp in the development of tissues and embryo remains largely unknown. Its role in HCC and several other types of human cancer indicate its potential role in the tumorigenesis in human cancer, a field remains to be explored. Genetically-engineered mice using transgenic technology and gene disruption (knockout) in mouse embryonic stem (ES) cells by homologous recombination to manipulate the mouse germ line may serve as ideal models for the study of development and oncogenesis. The unexpected results regarding ectopic expression, embryonic lethality and gene disruption with no phenotypes (null mutations) can be circumvented by modified techniques using retroviral vectors, and tissue specific and conditional knockouts. In light of the growing potential of transgenic and knockout mice in modern biomedical sciences, we use these techniques to study the potential crucial role of L2DTL/ramp in both embryogenesis and tumorigenesis of HCC and various other types of cancer, we would like to submit this proposal to study the gene. In addition to the creation of L2DTL/ramp transgenic mice, mice with disrupted L2DTL/ramp will be generated by traditional strategy as well as tissue-specific and conditional knockouts using the Cre/LoxP system. The specific aims are to create mice with over-expression and null-expression of the L2DTL/ramp gene by transgenic and knockout mice. The engineered mouse lines can be used for the following studies. Section 1: L2DTL/Ramp transgenic mice (1) To create transgenic mice which express human L2DTL/ramp gene in the livers by using liver-specific promoter. (2) The role of L2DTL/ramp in liver cancer is unknown. To study the role of L2DTL/ramp in liver cancer by using phenotype analysis and immunohistochemical and morphometric analyses which are dependent on the experimental results. (3) To elucidate the importance of L2DTL/ramp in the development of experimental HCC. Liver tumor will be induced in the L2DTL/ramp transgenic mice by chemical hepatocarcinogens.

5 Section 2: L2DTL/Ramp knockout mice (1) To verify whether L2DTL and Drosophila l(2)dtl are real homologues via the observation of embryonic lethality in homozygous knockout mice. Although the N' terminals that carries WD40 repeats are highly homologous between the two genes, the C' terminal of L2DTL has unique sequence not identified in Drosophila l2dtl and other human genes. (2) To study the role of L2DTL/ramp in cell growth, differentiation, and maturation in multiple tissues during embryogenesis. Gene targeting and knockout technologies will be used to generate mice with null expression of nuclear L2DTL/ramp.

6 ( 三 ). 報告內容 : Introduction: Hepatocellular carcinoma (HCC) is casually linked to chronic HBV and HCV infection, and the HBV related and non-hbv related HCCs differ in several important clinicopathological features (1), but the molecular mechanisms for HCC remain largely unknown. During the multistep tumorigenesis of human cancer, multiple genetic abnormalities accumulate and the tumor becomes more and more aggressive (2,3). These genetic alterations, critical to tumor progression, have been investigated intensively by various genetic techniques. The chromosomal alterations in HCC include various proportions of allelic loss and frequent gain at several chromosome regions (4-7). Gain of 1q is the most frequent change in HCC, involving 58-78% of the cases (4-7). Frequent gain of 1q is also observed in several other solid tumors (8,9). These observations indicate gain at 1q plays an important role in the tumorigenesis of HCC and other types of cancer. But, the putative genes with oncogenic potential remain to be identified. Using DD method to screen genes aberrantly expressed in HCC, we have successfully cloned several genes that are frequently overexpressed in HCC (10,11). In this study, we have identified a novel gene identical to human L2DTL (Genbank, AF195765). Human L2DTL is homologous to lethal (2) denticleless, l(2)dtl, initially identified in Drosophila melanogaster. Drosophila l(2)dtl is so designated because embryos with homozygous mutations of the gene lack ventral denticle belts and are lethal (12). Recently, L2DTL protein is shown to be a nuclear matrix-associated protein and is downregulated during RA-induced neuronal differentiation of NT2 cells, hence designated as ramp (13). L2DTL (ramp) is located at 1q (13), and has WD-40 repeats at the amino terminus. WD-40 repeats proteins are found in all eukaryotes but not in prokaryotes, and involved in several important biologic functions. These include cell division, cell-fate determination, gene transcription, transmembrane signaling, mrna modification and vesicle fusion (14-18). In addition to the WD-40 repeats domain, human L2DTL has a unique carboxyl terminus. L2DTL (ramp) is closely related to cell proliferation of NT2 cells (13). Despite these observations, the physiologic function of human L2DTL remains largely unknown, and its role in human cancer has not been studied. In our study, we found that L2DTL mrna was detected in multiple fetal tissues, but the level was very low in most adult tissues, including liver. L2DTL mrna was overexpressed in 53% of 271 unifocal primary HCCs, and associated with AFP elevation (P = ), grade II-IV HCC (P = ), early IHTR (P = ), and worse 10-year survival (P = ). To elucidate the role of L2DTL (ramp) in HCC, we correlated its mrna expression with clinicopathological features. L2DTL (ramp) mrna was overexpressed more often in HCCs of patients below average age of 55 years (P=0.0063), and in the patients with serum AFP elevation (P=0.0012). The overexpression also positively correlated with p53 mutation (P=0.0022), but did not correlate with gender, chronic hepatitis B and/or C virus infection, and Child-Pugh grade (data not shown). Histopathologically, L2DTL (ramp) mrna overexpression occurred more frequently in large HCC bigger than 3 cm, (P=0.0002), and in grade II to IV HCC than grade I HCC (P=0.0001). It was important to note that stage IIIA to IV HCC that had various extent of intrahepatic vascular invasion had L2DTL (ramp) mrna overexpression two-fold higher than low-stage (stages I and II) HCC, P= Hence, the developmentally regulated L2DTL plays an important role

7 during embryogenesis and the malignant transformation of hepatocytes. Its overexpression appeared to occur earlier than p53 mutation during the tumor growth of HCC, and might serve as an unfavorable prognostic factor. A combinatorial analysis of L2DTL overexpression and p53 mutation was useful to identify subsets of HCC with high risk of early IHTR and poor prognosis. L2DTL mrna was also overexpressed in several other common cancers. This may open new perspectives for gene therapy for HCC and other cancers. Although L2DTL is homologous to Drosophila l(2)dtl in the N' terminal that harbors the WD40 repeats, L2DTL has a unique sequence in its C' terminal. This sequence is not seen in Drosophila l(2)dtl, or in other human genes deposited in GenBank. Therefore, whether L2DTL is indeed a human homologue of Drosophila l(2)dtl remains to be verified. In view of the importance of Drosophila l(2)dtl in murine embryonic development, and L2DTL in human fetal tissues, it is very likely they are homologues. Because of its potential crucial roles in several common human cancers, particularly HCC, its roles in cell growth and maturation and tissue development warrant our efforts to delineate. Portal vein spread is the most devastating event for the poor prognosis of HCC, and its molecular mechanisms remain largely unknown. The close association of L2DTL mrna overexpression with tumor progression and metastasis of HCC gives us a chance to verify in transgenic and knockout animal models, and an opportunity to decipher the potential molecular mechanisms. Specific Aims: Hypothesis to be tested: 1. L2DTL is a human homologue of Drosophila l(2)dtl: Human L2DTL has a unique C' sequence not found in Drosophila l(2)dtl, in addition to the WD40 repeats are present in many genes in eukaryotes. The verification that the two genes are real homologous will have enormous impact in the study of its role not only in embryogenesis but also in evolution. 2. L2DTL is crucial for embryogenesis of multiple tissues in human embryonic development, its homozygous deletion may be lethal. If not, redundant molecules need to be identified. 3. L2DTL is crucial for liver regeneration and the tumorigenesis of HCC and other cancers. 4. L2DTL plays important role in cell migration and tumor metastasis. Objectives: Human L2DTL is homologous to lethal (2) denticleless, l(2)dtl, initially identified in Drosophila melanogaster. Drosophila l(2)dtl is so designated because embryos with homozygous mutations of the gene lack ventral denticle belts and are lethal. L2DTL is a WD-40 repeats nuclear protein, is also known as ramp. The specific aims are to study by transgenic and knockout mice: About transgenic mice: (1) To study the importance of L2DTL/ramp in the development of HCC. Liver tumor will be induced in the human L2DTL/ramp -transgenic mice by hepatocarcinogens. Tumor cells isolated from these mice will be injected into SCID mice, and the importance of L2DTL/ramp in tumor metastatic potential examined by pathological study. We plan to create mice with over-expression of the human L2DTL/ramp gene by tissue-specific targeting technology. The engineered mouse

8 lines can be used for the following studies. (a) phenotype analysis dependent on the experimental results (b) immunohistochemical and morphometric analysis. (2) To assess the importance of L2DTL/ramp during liver regeneration induced by chemical injury in transgenic mice. About knockout mice: (1) To study the role of L2DTL/ramp in cell growth, differentiation, and migration during embryogenesis. Gene targeting and knockout technologies will be used to generate mice with null expression of nuclear L2DTL/ramp. To circumvent problems of embryonic lethality due to whole body knockout, which occurs in Drosophila, conditional and tissue specific disruption of L2DTL/ramp will be applied. (2) To study the role of L2DTL/ramp in cell growth and maturation in multiple tissues during embryogenesis by knockout mice. Studies and Results : The main purpose of the project is to create L2DTL/ramp transgenic and knockout mice that may allow us to analyze L2DTL/ramp s functions and the consequences of the over-expression and disruption during the development of the mouse embryos to adulthood. Experimental procedures to achieve these goals are outlined and described in following sections. Section 1. Human L2DTL/ramp transgenic mice. (1) Construction of human L2DTL/ramp mini-gene by using liver specific promoters Transgenic technology will be performed by manual injection of DNA into the pronuclei of mouse zygotes. Microinjection, implantation and breeding of the transgenes will be carried out as described (19). The pronuclei from FVB, C57BL/6, and C57/CBA strain will be used because of their large pronuclei, transparent cytoplasm and easy to obtain by superovulation (19). The cdna coding of human L2DTL/ramp obtained by RT-PCR using the hepatoma mrna as the template was cloned into the pcmvtag2b vector (20), and used to construct expression plasmids. We used the palb.polya vector (fig 1) for construction (21) so that the cdna was under the control of the Albumin enhancer/promotor for specific expression in the liver. Another expression plasmid was contructed into pbs-hcri-a vector (fig 1). This plasmid containing the apolipoprotein E locus control region (HCR), human α1-antitrypsin (haat) promoter, a portion of the first intron (intron A) of human FIX (hfix intron A), and a bovine growth hormone polyadenylation signal (bghpa). The presence of intron A can enhance FIX expression in mice liver (22). The resultant plasmids were linearized before the injection into the zygotes.

9 Fig 1. The vectors used for construction of human L2DTL/ramp transgene (2) The results of L2DTL/ramp Transgene construction Utilizing the 2.3 kb coding region of human-l2dtl cdna, we had generated two transgenic targeting vectors with different liver specific promoters: Albumin and human α1-antitrypsin (haat). The main framework of the constructs comes from palb-polya and pbs-hcrhpi-a, respectively. In order to get the stable expression in mice, we inserted the 1.5 kb long intron 2 of human-l2dtl obtained by PCR-fusion, into human-l2dtl 2.3 kb coding region cdna. In addition, a Flag-tag, serving as a tool in detecting protein expression, was inserted into the 5 end of the full-length human-l2dtl cdna. Insulators are regulatory elements that establish independent domains of transcriptional activity within eukaryotic genomes. Insulators possess two properties: an anti-enhancer activity that blocks enhancer promoter communication, and an anti-silencer activity that prevents the spread of repressive chromatin (23). In order to control the expression level in mice, we inserted two copies of insulators in the palb-flag-l2dtl-i2 Tg vector in front of the Albumin promoter. Finally, two transgenic targeting vectors (figure 2) PBS-Flag-L2DTL-I2 Tg and Insulators-pAlb-Flag-L2DTL-I2 Tg were obtained. Fig 2: The L2DTL-Tg Targeting vectors. Both constructs contain liver specific promoter, Flag-tag, full length human-l2dtl cdna and intron 2. The palb.polya construct contains two copies of insulators in front of the Albumin promoter. (3). Cell lines transfection to verified the protein expression from these two transgenic

10 constructs. These two L2DTL-Tg Targeting vectors were transfected into the hepatoma cell line (Huh7 cell) and cervical cancer cell line (HeLa cell). The L2DTL/ramp protein expression was verified by the immunohistochemical staining with anti-flag antibody. HeLa and Huh-7 cells were plated in 6-well culture plate with coverslip at density of 2 X 10 5 cells per well and transfected with the L2DTL transgene plasmid at a final concentration of 1 μg of DNA per well. Transfections were performed with the lipofectamine 2000 reagent (INVITROGEN, life technology, USA). Cells were cultured on coverslip in 6 well culture dish for 24 hours, and then fixed with 4% paraformaldyhide 10 min at room temperature, followed by 1XPBS rinse for 3 times and incubation in PBST(0.2% Triton X-100 in PBS)for 10 min, fixed with 0.3 % H 2 O 2 in methanol for 10 min, 1XPBS wash 2 times, and blocking with 5% FBS in PBST for 30 min. Add primary antibody (anti-flag antibody,10 ug/ul in 5% FBS, Sigma.) and incubate for 2 hours at room temperature. Wash with PBST for 3 times, 5 min each. Then add secondary Ab (Histonfine ready-use) for 30 min. Wash with PBST for two times, 5 min each. Then use DAKO Liquid DAB+ substrate- chromogen System (DAKO, Glostrup, Denmark) for color development, counter stain by hematoxylin, and mount with permount media. Results as the fellow figure 3 and figure 4. PBS-Flag-L2DTL-I2 Tg (cell line transfection) HeLa cell, 200X. HeLa cell, 400X. Huh-7cell, 200X. Huh-7cell, 400X. Fig 3. Transfection with PBS-Flag-L2DTL-I2 Tg plasmid construct in HeLa and Huh-7 cell lines. The L2DTL protein is expressed in the nucleus of Huh-7(hepatoma cell line) cell, but not in HeLa (cervical cancer cell line) cell.

11 Insulator-Alb-Flag-L2DTL-I2 Tg ( cell line transfection) HeLa cell, 200X. HeLa cell, 400X. Huh-7cell, 200X. Huh-7cell, 400X. Fig 4. Transfection with Insulator-Alb-Flag-L2DTL-I2 Tg plasmid construct in HeLa and Huh-7 cell lines. The L2DTL protein is expressed in the nucleus of Huh-7 (hepatoma cell line) and in HeLa (cervical cancer cell line) cells. Section 2: L2DTL/ramp knockout mice (1). Screening and analysis of mouse BAC clones We used human L2DTL cdna as probe to screen mouse gemonic DNA library (high density mouse BAC colony membrane, ResGen) and isolated 4 positive BAC clones: clones D ~ G. Digestion of BAC clone DNA with NotI showed two bands; the high molecular weight band was mouse genomic DNA insert (Fig. 5) and the 6.8 kb band vector DNA. Through the collaboration with Celera via Vita Genomics, we obtained partial sequence of mouse L2DTL gene including exons 1, 3-7, 14 and flanking intron sequqences. To confirm these BAC clones contain L2DTL gene, we designed 3 pairs of primers to amplify exons 1, 3 and 14. We found that clones D ~ F contained L2DTL exons 1 and 3, and clone G contained all these 3 exons (data not shown).

12 Fig 5. Genomic DNA insert of each BAC clone. Clones D and F: 110 kb, clone E: 55 kb, clone G: 145kb. M: marker. (2). Subcloning of DNA fragment contain exons 1-5 by gap repair Recently, a highly efficient recombination system for chromosome engineering in E. coli has been described. A defective lamda prophage was used to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence. It is important that the recombination is proficient with DNA homologies as short as bp, making it possible to use PCR-amplified fragments as the targeting cassette. Using this system, subcloning of DNA fragments as large as 80 kb from BACs can be performed by gap repair, obviating the need for restriction enzymes or DNA ligases. We designed two chimeric primers for PCR amplification of linear plasmid vector. The 5 51 nts of each primer was homologous to the two ends of the BAC sequence to be subcloned, the 3 20 nts are homologous to PBR322. Subcloning relies on gap repair to recombine the free ends of the linear plasmid vector with homologous sequences carried on the BAC. Recombination generates a circulate plasmid in which the DNA insert was retrieved from the BAC DNA. Using this system, we had successfully subcloned 10 kb fragment containing exons 1 5 of L2DTL (Fig. 6) by gap repair. Sequencing analysis showed this DNA fragment (named 1D5U) containing exon 2 in the 561 bp gap which was not presented in the Celera database. Digestion of 1D5U with different REs showed pattern as predicted (Fig. 7). We also sequenced the 5 and 3 ends of 1D5U to confirm the right sequence (data not shown). The next step will be to construct targeting vector base on this DNA fragment. Each primer also contains 20-nt segments at its 3 end to primer pbr322. NotI and SalI cleavage sites were included in these primers to facilitate release of the subcloned fragments from the plasmid backbone.

13 Fig 6. Fig 7. Fig. 6. Subcloning of exons 1 to 5 of L2DTL from BAC clone by gap repair. Gap repair intermediate showing pairing between the amplified pbr322 targeting cassette and the BAC. The locations of the 5 homologies on the amplification primers used to amplify pbr322 for subcloning by gap repair are shown as thick black arrows. amp, amp-resistance gene; ORI, origin of replication. Fig. 7. Digestion of 1D5U DNA by SacI, PmeI, NaeI and BstEII. These REs cut out five fragments as predicted (3.8, 3.2, 2.4, 2.1, and 1.0 kb). (3). Making 2 targeting constructs, containing distinctive selection markers Neo/tk and HPRT/tk. The unwanted results regarding ectopic expression, embryonic lethality and gene disruption with no phenotypes (null mutations) can be circumvented by modified techniques using tissue specific and conditional knockouts. The mice with disrupted L2DTL/ramp are to be generated by traditional strategy as well as tissue-specific and conditional knockouts using the Cre/LoxP system. Strategy for constructing the targeting vector based on pko-loxp (neo and tk) and ppnt-hprt-loxp (hprt and tk) is depicted in (Figure 8). Briefly, to obtain a third loxp, we used the pulwl vector to generate a 6.8 kb 5 large L2DTL-loxP fragment with a loxp within. The 3' small fragment (PmlI-BstXI;1.9 kb) was inserted into the pko-loxp targeting vector at NsiI site, while the large fragment ( 8.6 kb) from pulwl-l2dtl was subsequently inserted into 3 L2DTL- pko-loxp targeting vector at XhoI site. Then, the selection marker, Neo was substituted by HPRT and thus the second targeting vector was obtained. Finally, both constructs (Figure 9) were linearlized by AscI digestion. In the first year of this project, the constuct procession has been completed. These two L2DTL- conditional KO targeting vectors as show in the figure 9 had been transfected into the ES cells, and the stable ES cell lines with L2DTL-KO targeting vectors were selected by the Neo or HPRT selection marker.

14 Fig 8. Cloning strategy of 1D5U-L2DTL to pko-loxp. The wild type 1D5U-L2DTL is 10 kb in length which with 5 exons in it. The 3' fragment is 1.9 kb and the 5' fragment is 6.9 kb with a 3th loxp in it. Fig 9. The conditional knockout targeting vectors. Exon2/Exon3 region about 1.7kb long in each construct is flanked by 2 loxp sites.the selection markers are Neo-tk and HPRT-tk respectively.

15 Section III: The results of L2DTL/ramp antibodies production. The coding region of L2DTL (ramp) was subcloned to the expression vector pegfp-c1 in Xho I and BamH I sites (Clontech, Palo Alto, CA, USA). The sequence was verified. The 293T cells were transfected with the L2DTL-pEGFP-C1 plasmid using the lipofectamine 2000 reagent (Invitrogen, life technology, USA). After transfection for 24 hours, the cells were fixed in 4% paraformaldehyde, and stained with hoechst (0.2 g/ml), and observed under fluorescence microscopy. For Western blotting, 293T cells were transfected with L2DTL-pEGFP-C1 and harvested 24 hours later. Protein samples (60 g) were separated in 10% SDS-PAGE gel, electrotransferred to nitrocellulose membrane (Amersham Pharmacia Biotech, UK), and then incubated with anti-egfp at 1:400 (Clontech, Pald Alto, CA, USA), or anti- L2DTL (ramp) polyclonal antibody generated against synthetic peptides derived from the N termini of L2DTL amino acid sequence at 1:500. Membranes were then incubated with anti-rabbit IgG HRP-conjugated secondary antibodies (1:10000, Roche Molecular Biochemicals, Germany), and immunoreactive bands were detected using an ECL kit (Amersham Pharmacia Biotech, UK) (figure 10). Fig. 10. (A) Amino acid sequence of human L2DTL (ramp) of 730 amino acids. The five WD-40 domains in the amino terminus are shaded, and the nuclear localization signal is boxed. The leucine zipper motif was underlined and the SH3-binding consensus in the carboxy terminus double underlined. (B) Subcellular localization of EGFP-L2DTL. EGFP-L2DTL fusion protein was expressed in fine dots in nuclei (left), as shown by Hoechst stain (right). (C) Western blotting. The EGFP-L2DTL fusion protein of approximately 113 kda was recognized by anti-egfp (Lane 2) and anti-l2dtl antibodies (Lane 4). Lanes 1 and 3, pegfp-c1 vector transfected; Lanes 2 and 4, L2DTL-pEGFP-C1 vector transfected.

16 Discussion: L2DTL/ramp is a good candidate for transgenic and knockout experiments not only because it plays important role in human embryogenesis, but also plays important roles in the tumorigenesis of several common human cancers, particularly metastatic potential of hepatocellular carcinoma. To our knowledge, murine L2DTL/ramp homologue has not been reported, and we may be the first to conduct this study. The main purpose of this project is to create L2DTL/ramp transgenic and knockout mice that may allow us to analyze L2DTL/ramp s functions and the consequences of its disruption during the development of the mouse embryos to adulthood. Methods and strategies for investigating L2DTL/ramp s role in cell growth, migration and tumor metastasis are discussed in section I. Strategies for creating conditional and tissue specific knockouts are described in section II. Since it is important to have anti- L2DTL/ramp antibodies, we had synthesized a peptide of 24 amino acids (from the N termini of L2DTL/ramp amino acids sequence, 186-DGAHNTSDKQTPSKPKKKQNSKG -208) for immunization. The work of knock-out animal model was carried out at the lab of co-investigator, Prof. Shu-Hwa Lin. Section I: human L2DTL/ramp transgenic mice (1). About transient transfect results of the two transgenic constructs The two transgenic constructs had transient transfection to Huh-7 and HeLa cells. The results are described in fig.3 and fig.4. The PBS-Flag-L2DTL-I2 Tg construct has a human α1-antitrypsin promoter and specific expression of human L2DTL protein in the nuclei in liver cells only, such as Huh-7 hepatoma cell line. As for another construct named Insulator-Alb-Flag-L2DTL-I2 transgenic vector a albumin promoter. Although various transgenic mouse models have reported that the albumin is a liver specific promoter, human L2DTL protein was not only expressed in the Huh-7 hepatoma cell line but also in the HeLa cervical cancer cell line. (2). Strategies for analyzing L2DTL/ramp's functions by L2DTL-Tg mice. (a) DNA and RNA analyses of L2DTL/ramp transgenic mice. General methods of isolation and quantitation of genomic DNA from mouse tails, as well as detection of RNA from individual organs were done as described (11). Tissue-specific distribution of the transgenes was performed by Southern blotting, and in situ hybridization. Northern blotting will be used to detect the expression level of human L2DTL/ramp in transgenic mice. We are aware of the necessity in identifying the integration site of the L2DTL/ramp in the transgenes, the procedures had been described (24). (b) Histological and immunohistochemical experiments and protein detection. General techniques had been described (1), For protein detection, the antibody against L2DTL/ramp will be a very useful tool. Some L2DTL/ramp polyclonal antibodies had been obtained (as described fig 10 and below). Western blotting was done on the standard procedures (25). For histological investigations, paraffin-embedded tissue sections will be prepared by fixing them in 10% buffered neutral formalin and then getting processed (26). Fixed specimens are embedded with glyco methacrylate (GMA) and um sections will be made and stained with a modified haematoxylin and eosin procedure.

17 (c) Morphological analysis of the transgenic mice. We will analyze the pathological changes of liver tissue and the life span of the transgenic mice with that of wild type. Provided with the results of phenotype manifestations in these transgenic mice, other possible functions of L2DTL/ramp could be revealed. (d) Tumor induction in L2DTL/ramp Tg mice. If the transgenic mice with overexpressed L2DTL/ramp in liver have no significant tumor formation, we will carry out carcinogen injection into L2DTL/ramp Tg mice and wild type mice aiming at inducing hepatoma in these mice. The physiological condition of the Tg mice after DEN treatment will be followed up each month. An assumption is made such that the liver tissue of the Tg mice should be different from that of the wild-type. Monitoring tumor formation in these animals would be helpful in characterizing the role of L2DTL/ramp in tumorgenesis. Section II: L2DTL/ramp knockout mice (1). About the construct of conditional KO targeting vectors In the conditional knockout targeting vectors, Exon2/Exon3 region about 1.7kb long (named as short arm) in each construct is flanked by 2 loxp sites. In front of the first loxp site was the long arm of 6.8 kb in length (named as long arm). According to our experience, the portion of the short arm to the long arm was 1:3 that would be used successfully in ES cell targeting. One of the targeting vector with HPRT selection marker is reverse type in loxp sites. It will reverse the Exon2/Exon3 region of mouse L2DTL gene, which will out of frame of mouse L2DTL gene. (2). Strategies for analyzing L2DTL/ramp's functions by L2DTL-KO mice. (a) The role of L2DTL/ramp in embryonic development. The targeted ES (embryonic stem) cell clones will be determined by positive and negative selection while exon 2 and 3 of L2DTL, which were flanked by 2 loxps will be deleted by exogenously introduced cre recombinase and thus hemizygous L2DTL-KO ES clones are obtained. A second targeting of the hemizygous L2DTL-KO ES cell using similar approach mentioned above will yield homozygous L2DTL-KO ES clones. Subsequently, these ES cell clones are microinjected into mouse blastocysts to generate L2DTL-KO mice. During the course of development, pregnant female mice bearing embryos at different stages are sacrificed and their embryos will be examined by H/E stain. If L2DTL is really important in the development of mouse, embryonic lethality or abnormal development of the embryos could be expected. (b) The role of L2DTL/ramp in cell growth. Through the observations of the gross anatomy (body and organ weights and sizes), histology (cell size and contents), and phenotypes of the chimeric, f1 heterozygous, and f2 homozygous knockout mice as wells as their embryos, the function of L2DTL/ramp in cell growth and morphology and embryogenesis may be determined. Normal mice of same age and gender will be used as controls. We are aware that no phenotypic change may happen to knockouts (gene redundancy). We also aware that homozygous mutation of L2DTL/ramp may lead to embryonic lethality, because Drosophila embryos lack of l(2)dtl are lethal. In case this will happen to L2DTL/ramp knockout, overexpression by transgenic technology will be an alternative approach to study. (c) The role of L2DTL/ramp in spontaneous tumor development. The targeted ES cell clones will be microinjected into mouse blastocysts to generate

18 chimeras and then be germline transmitted into f1 heterozygous and gradually f2 homozygous targeted mice. Crossing Albumin-Cre tg mice with f2 mice will give birth to the L2DTL-liver specific KO mice. Through comparison among the phenotypes of the chimeras, f1 heterozygous, and f2 homozygous KO mice as wells as their embryos, based on gross anatomy observations (body and organ weights and sizes) and histological dissection (cell size and contents), the function of L2DTL/ramp in cell growth and morphology could be revealed. (d) The role of L2DTL/ramp in tumor growth, invasion, and metastasis. The expression of L2DTL/ramp in ES cells is to be proved as soon as the antisera are available. We have successfully used RT-PCR analysis to check the presence of L2DTL/ramp mrna in several human cancer tissues especially hepatocellular carcinoma. RT-PCR will also be used to measure the gene expression in ES cells. The KO ES cells and their in-vitro differentiated fibroblast or epithelial cells will be assayed for their in-vitro growth rates in comparison with their wild-type and heterozygous counterparts. The invasive/metastatic potential of tumor cells with overexpression or lack of expression of L2DTL/ramp will be tested. To obtain HCC cells, we plan to induce HCCs in normal and L2DTL/ramp KO mice using hepatocarcinogens, such as DEN (Diethylnitrosamine) (27). The physiological condition of of the KO mice after DEN treatment will be followed up each month. It is assumed that the induced tumor cells in these mice should show difference from those of the wild-type mice. The method of the induction of HCC in mice is as described previously (28). These induced tumor cells from KO and wild type will be injected into SCID mice to evaluate the potential of tumor growth and metastasis, concerning the number and size of liver tumor nodules, the morphology and intrahepatic portal vein spreading and distant metastases. All of the above features will be characterized by autoptic examination of the mice. The results will be compared with those derived from injected primary HCC cells. Section III: Production of antibodies. We plan to express the human L2DTL/ramp cdna in the T7 phage system (29) to obtain fulllength protein for immunization. We will use the pet15b vector to express L2DTL/ramp protein in the E. coli BL21(DE3)/pLysS strain. The protein will be purified (30) and used to raise polyclonal antibody. Since mouse and human share high homology of amino acid sequence identity, rabbit polyclonal antibodies should be easier to produce than mouse monoclonal antibodies. Immunization will be carried according to standard procedure. We also synthesized a peptide of 24 amino acides (from the N termini of L2DTL/ramp amino acids sequence, 186-DGAHNTSDKQTPSKPKKKQNSKG-208) that is unique for L2DTL/ramp for immunization (see the fig. 10. results).

19 Reference: 1. Hsu, H.C., Jeng, Y.M., Mao, T.L., Chu, J.S., Lai, P.L., and Peng, S.Y. -Catenin mutations are associated with a subset of low stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis. Am. J. Pathol., 157: , Sager R. Tumor suppressor genes: the puzzle and the promise. Science 246: , Aaroson, S.A. Growth factors and cancer. Science 254: , Marchio, A., Meddeb, M., Pineau, P., Danglot, G., Tiollais, P., Berheim, A., and Dejean, A. Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosom. Cancer, 18: 59-65, Kusano, N., Shiraishi, K., Kubo, K., Oga, A., Okita, K., and Sasaki, K. Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinoma: their relationship to clinicopathological features. Hepatology, 29: , Wong, N., Lai, P., Lee, S.W., Fan, S., Pang, E., Liew, C.T., Sheng, Z., Lau, J.W., and Johnson, P.J. Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis. Am. J. Pathol., 154: 37-43, Guan, X.Y., Fang, Y., Sham, J.S.T., Kwong, D.L.W., Zhang, Y., Liang, Q., Li, H., Zhou, H., and Trent, J.M. Recurrent chromosomal alterations in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosom. Cancer, 29: , Simon, R., Burger, H., Brinkschmidr, C., Bocker, W., Hertle, L., and Terope, H.J. Chromosomal aberrations associated with invasion in papillary superficial bladder cancer. J Pathol., 185: , Tirkkonen, M., Tanner, M., Karhu, R., Kallioniemi, A., Isola, J., and Kallioniemi O.P. Molecular cytogenetics of primary breast cancer by CGH. Genes Chromosomes Cancer, 21: , Huang, L.R., and Hsu, H.C. Cloning and expression of CD24 gene in human hepatocellular carcinoma: A potential early tumor marker gene correlates with p53 mutation tumor differentiation. Cancer Res., 55: , Hsu, H.C., Cheng, W., and Lai, P.L. Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular caricnoma: Biological significance and temporospatial distribution. Cancer Res., 57: , Kurzik-Dumke, U., Neubauer, M., and Debes, A. Identification of a novel Drosophila melanogaster heat-shock gene, lethal (2) denticleless [l(2)dtl ], coding for an 83-kDa protein. Gene, 171: , Cheung, W.M.W., Chu, A.H., Chu, P.W.K., and Ip, N.Y. Cloning and expression of a novel nuclear matrix-associated protein that is regulated during the retinoic acid-induced neuronal differentiation. J. Biol. Chem., 276: , Yochem, J., and Byers, B. Structural comparison of the yeast cell division cycle gene CDC40 and a related pseudogene. J. Mol. Biol., 195: , Choi, W.J., Clark, M.W., Chen, J.X., and Jong, A.Y. The CDC4 gene product is associated with the yeast nuclear skeleton. Biochem. Biophys. Res. Commun., 172: , Simon, M.I., Strathmann, M.P., Gautam, N. Diversity of G proteins in signal transduction. Science, 252: , van der Voorn, L., and Ploegh, H.L. The WD-40 repeat. FEBS Lett., 307: , Neer, E.J., Schmidt, C.J., Nambudripad, R., and Smith, T.F. The ancient regulatory-protein family of WD-repeat proteins. Nature, 371: , Hogan, B., Beddington, R., Costantini, F., Lacy, E. Manipulating the mouse embryo. 2nd Edition, SH Laboratory Press, Schutte, B. C., Ranade, K., Pruessner, J., Dracopoli, N. Optimized conditions for cloning PCR products into an XcmI T-vector. Biotechniques. 22, 40, Guilhot, S., Huang, S. N., Xia, Y. P. La Monica, N., Lai, M. M., Chisari, F. V. Expression of the hepatitis delta virus large and small antigens in transgenic mice. J Virol. 68, 1052,

20 Miao, C. H., Ohashi, K., Patijn, G. A., Meuse, L., Ye, X., Thompson, A. R. Kay, M. A. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol. Ther. 1, 522, Kuhn, E. J., Geyer, P. K. Genomic insulators: connecting properties to mechanism. Current Opinion in Cell Biology: 15, 259, Hogan B, Beddington R, Costantini F, and Lacy E. Manipulating the mouse embryo. 2 nd Edition, CSH Laboratory Press, Lin, S. W., Lin, C. N., Hamaguchi, N., Smith, K. J., and Shen MC. Characterization of a factor IX variant with a glycine207 to glutamic acid mutation. Blood 84, 1866, Erikson, P. A., Lewis, G. P., Fisher, S. K. (1993). Postembedding immunocytochemical techniques for light and electronmicroscopy. In Antibodies in cell biology. (ed. Asai DJ) (Academic Press Inc, San Diego) 27. Tsukamoto, T., Inada, K., Fukami, H., Yamamoto, M. Mouse strain susceptibility to diethylnitrosamine induced hepatocarcinogenesis is cell autonomous whereas sex-susceptibility is due to the micro-environment: Analysis with C3H BALB/c sexually chimeric mice. Jpn.J.Cancer Res. 91: , Hanigan MH, Winkler ML, Drinkwater NR. Induction of three histochemically distinct populations of hepatic foci in C57BL/6J mice. Carcinogenesis 14: , Studier, W.A., Rosenberg, F., Dunn, J., Dubendorff, J. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymology 185, 60-88, Huang, C. C., Li, L. T., Shen, M. C. Chen, J. Y., Lin, S. W. Domain specific monoclonal anti-factor VIII antibodies generated by inclusion body-renatured factor VIII peptides. Thromb Resea. 101, 405, 2001.