Abstract
BACKGROUND: Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells.
OBJECTIVE: To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP16 gene.
DESIGN: Constructing an inserting inactivation target by inserting SA-RIES-βgeo expression cassette.
SETTING: Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA.
MATERIALS: The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from Invitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen Biotech Corp. pcDNA3.1(+) vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University.
METHODS: The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction (PCR) screening. The SA-RIES-βgeo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis.
MAIN OUTCOME MEASURES: Clones with homologous recombination.
RESULTS: The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES-βgeo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted.
CONCLUSION: A LRP16 gene targeting vector is constructed and a homologous recombinant is obtained.

INTRODUCTION

LRP16, a human gene that was originally recognized and isolated in 1999 by our group, is identified to be an estrogen responsive gene[1-5]. Its expression level is dependent on the estrogen activity in estrogen ERα positive breast cancer cells. Recent evidence has demonstrated that LRP16 is a coactivator of ERα[6,7]. Our previous studies have identified a key cis-element within the promoter region of LRP16 gene responsive to 17β-estrodiol (E2) [8] and demonstrated that the overexpression of LRP16 gene promotes the proliferation and invasive growth of breast cancer cells [5-7]. Sequence analysis shows a conserved domain in the carboxyl terminal of LRP16, which have been implied to be very important to maintain the vital movement of the cells. To investigate the biological function of LRP16 at the level of an intact animal, a LRP16 gene targeting vector was constructed by insetting the SA-RIES-βgeo into the fifth exon of mouse LRP16 gene, and was transfected into the mouse embryonic stem cells (ES cells). An ES cell clone with target sequence homologously inserted was identified, which is necessary for establishing an LRP16 knock-out mouse strain and for further study on the biological function of the LRP16 gene in vivo.
MATERIALS AND METHODS

Materials
Plasmid, cell strain, PCR and sequencing primer: pBluescript SKⅡ+ and mouse genomic library in pBeloBAC11 Vector were purchased from Invitrogen Corp., TopF10 competent cells were purchased from Beijing Tiangen Biotech Corp., pCDNA3.1(+) vector was kept in our laboratory. Mouse embryonic stem cells were kindly provided by Dr. Masatoshi Nomura at Kyushu University of Japan. Primer sets of P1 (5'-GCA CCC TAC AGA ACT GCG AGA C-3') and P2 (5'-GGC TGA AGG AAA GGA CAG AAC C-3') were used for the genomic library screening; P3 (5'-CCA CGG AAA CAA TCA CAG A-3') and P4 (5'-CGG GCA GAT AGA AAG GAG-3'), and P5 (5'-AGG CCA GGC GTA TTT GTT GTG AGA-3') and P6 (5'-GAG CGG GTG TTA GAG GTC CAG GTT-3') for preparing the probes of 5'-terminal and 3'-terminal southern blot, receptively.
Enzyme and other reagents: Restriction enzymes, alkali phosphatase (CIAP) and Taq enzyme were purchased from Takara Corp.; T4 DNA ligation Kit and probe labelling Kit from Promega Corp.; Plasmid DNA purification system, Genomic DNA Purification system, Large-Construct Kit and Gel Extraction Kit from QIAGEN Corp.; DMEM

medium,non-essential amino acid, 0.5% trypsinase (1:250) and fetal bovine serum (FBS) from Gibico Crop.; Penicillin, streptomycin, mitomycin, LIF, G418 and collagen from Sigma Crop.; PBS, Southern trarsmembrane, and prehybridization solution were prepared by our laboratory; [-P32]dCTP was purchased from YaHui Chemical Corp.

Methods
The experiment was conducted in the Department of Molecular Biology, the General Hospital of Chinese PLA and the Bioregulatory Laboratory in Kyushu University form November 2004 to May 2005.

Genomic library screening and targeting fragment subcloning
100 clones from the mouse genomic library were picked out from LB flat plates containing amphemycin and were screened by PCR analysis using P1 and P2 primer sets. The reaction parameter of PCR was 95℃ for 5 minutes (pre-denaturing); 30 cycles of 94℃ for 30 seconds (denaturing), 60 ℃ for 30 seconds (annealing) and 72 ℃ for 1 minute (extension) and then 1 cycle of 72 ℃ for 10 minutes (extension). All PCR products were analyzed after electrophoresis in 0.8% agarose gel and the LRP16 containing clones were sequenced based on LRP16 genome sequence in Genbank. Finally, one positive clone, named as pBeloBAC11-LRP16, containing the sequence spanning 5'- and 3'-terninal region of the LRP16 gene was obtained. This plasmid was purified with a Large-Construct Kit according to the handbook and were digested using Hind Ⅲ. As expected, a 13.6-kb DNA fragment spanning from the partial intro 3 to intro 10 was got after Hind Ⅲ treatment, and was extracted from the gel, then was cloned into the pcDNA3.1(+) vector at Hind III sites. The ligation product using 13.6 kb fragment was transformed into the TopF10 competent cells. 100 colonies from the transformation products were picked out for insertion verification using Hind Ⅲ. Finally, one clone with 13.6 kb fragment insertion was got and was termed as pC18.

Strategy for gene knockout
One unrelated fragment was firstly inserted into the fifth exon of the mouse LRP16 gene at the SacⅡsite to introduce two SalⅠsites. Then the unrelated fragment was replaced by SA-IRES-βgeo using SalⅠenzyme. The SA-IRES-βgeo cassette was comprised of lucZ, neo, SV40 and ribosome insertion site and was frequently used in gene targeting manipulation [9] (Figure 1).

Construction and identification of the LRP16 targeting vector
The pC18 plasmid was firstly digested with Hind Ⅲ and EcoR V, then the Hind Ⅲ/EcoR V fragment was extracted and was again subcloned into pcDNA3.1(+) vector, and transformed into TopF10 competent cells. The product was named as pC18HEV. A 3 kb fragment from the 3'- terminal genomic DNA of the LRP16 gene was inserted into the pC18HEV vector using PCR-introduced SacⅡsites (named pC18HEVΔLRP16), meanwhile the Sal Ⅰ sites were also introduced into pC18HEV. The pC18HEVΔLRP16 vector was digested with XhoⅠand the target fragment was transferred to XhoⅠ/SalⅠ linearized pBluescript SKⅡ(+) vector. This recombinant was named pBlueΔLRP16. Because XhoⅠand SalⅠare isocaudarners, the site digested with them can be destroyed after ligation. SA-IRES-βgeo was inserted into the pBlueΔLRP16 at the SalⅠsites and the product was identified for the orientation of the SA-IRES-βgeo insertion by several enzymes. The correct one was named as pBΔ16 (Figure 2). The pBΔ16 targeting vector was further confirmed by EcoRⅠand BamHⅠ(Figure 3).

ES cells transfection and clone screening
Mouse ES cells were cultured on primary embryonic fibroblast cells (EMFI), which were isolated from 14-17 days mice embryo and were treated with mitomycin (100 g/L) to inhibit growth. LIF (1×105 U/L) was supplemented to DMEM medium to inhibit ES cell differentiation. All animal procedure in the experiment was strictly manipulated according to the International Animal Procedure Criteria. ES cells were transfected with linearized targeting vector DNA by electroporation and were screened with 200 mg/L G418. The G418-resistant ES cell clones were picked out and further cultured for genotypic analysis. ES cell genomic DNA was extracted for Southern blot identification.

Southern blot analysis
The 3'- and 5'- Southern blot assays were used for ES cell genomic DNA analysis. Briefly, the genomic DNA was digested with Hind Ⅲ. The 3'- terminal probe (485 bp) was amplified with P5 and P6 primer sets, and the 5'-terminal probe (1192 bp) with P3 and P4. The sites of digestion and the probe binding region were indicated in Figure 1. The total enzyme reaction system included 80 μL ES genomic DNA, 10 μL 10×M Buffer, and 10 μl Hind Ⅲ. The digestion products were electrophoresed in a 1% agarose gel at 30 V and were then transferred to nitrocellulose filter (NC filter) using siphon transferring method. The NC filter was probed for either 5'- or 3'-probe, pre-hybridized for 3 hours at 65 ℃, washed twice (15 minutes once), then was used for radioautography.

RESULTS

Identification for the orientation of SA-IRES-βgeo cassette
Firstly, pBΔ16 was identified whether SA-IRES-βgeo was inserted in by SalⅠenzyme pattern analysis, which would produce two expected bands with different sizes (10 690 bp and 6 783 bp). Subsequently, the vector was digested with BglⅡ to detect the insertion orientation. If the insertion was correct, there would be four different bands: 11 529 bp, 3 672 bp, 2 191 bp, and 83 bp. If the insertion was in reverse orientation, the bands would be 8 483 bp, 5 237 bp, 3 672 bp and 83 bp. The enzyme pattern analyses showed that the 31st and 40th clones were inserted correctly (Figure 4).

Identification of zymogram of pBΔ16
pBΔ16 was consisted of the following sequence regions: LRP16 genomic spanning from the fifth to the eleventh exon, positive selection gene SA-IRES-βgeo and backbone of pBluescript SKⅡ(+). The major enzyme sites were marked in Figure 3. As shown in Figure 5, pBΔ16 was digested to 5 expected bands (9 195 bp, 3 594 bp, 2 268 bp, 1 865 bp, 531 bp) by EcoR Ⅰ. The 31st and 40th clones were expected. In addition, multiple enzymes such as BamH Ⅰ were used for further confirming the targeting vector.

ES cells gene transfer and clone
The pBΔ16 plasmid DNA was linearized by Xho Ⅰ, purified by phenol, chloroform extracted, solved in PBS and stored at –80 ℃ for 2 days. ES cells that grew well and less differentiated were selected and introduced to targeting vector by electroporation at 300 V, 250 μLF (25 μg DNA, 8×106 ES cells). The electroporated cells were plated in DMEM media supplemented with LIF (1×106 U/L). After one-day culture, G418 (200 mg/L) was added for selection. One week after transfection, the living ES cell colonies were picked out using 20 μL microinjector into 96-well plate under microscope. A part of ES cells in wells were frozen to liquid nitrogen, and the remaining was used for genomic DNA extraction.

Southern blot analysis
Genomic DNA samples extracted from 100 ES clones with G418 resistance were subjected to Southern blot analysis with both 3' terminal and 5' terminal probes. By Southern blot screening using 3'-probe, two expected bands (13.6 kb and 11.5 kb) was only examined in the 49th ES cell clone. Based on this, southern blot analysis was also performed using 5'-probe in the 49th clone, the 7th and the 94th clones were used as negative control. As illustrated in Figure 6, two expected bands (13.6 kb and 8.5 kb) were examined in the 49th clone, but not in the controls. These results demonstrated that the LRP16 target vector was homologously recombinated in the 49th clone.

DISCUSSION

Gene targeting is a new molecular biology technology that developed in the past decades. It is widely used in gene function and disease research[9-12]. Although the detailed molecule mechanism involved in this process has not been elucidated, it has played an important role in the functional study on a definite gene. By this technology, the wild-type genomic sequence of the target cells can be replaced by the exogenous DNA through homologous recombination to change the inherited characters of cells at the entirely level. The alterations of the subsequent phenotype can help us to exactly define the gene function.
LRP16, a human gene that was originally recognized in 1999, has high expression level in testicle, ovaries and mucosa of colon, moderate level in prostate, small intestine, spleen and thymus, and low in peripheral blood leucocytes[2-3]. Based on cell-culture assays, we previously have recognized the E2 responsiveness of the LRP16 gene, and meanwhile we also observed that the expression level of LRP16 gene was strongly dependent on the estrogen activities in ERα-positive breast cancer cells[8, 13]. Moreover, previous studies have demonstrated that the LRP16 overexpression markedly promoted the proliferation of MCF-7 human breast cancer cells by promoting G1/S transition through increasing the cyclin E and cyclin D1 protein level [4, 6]. In contrary, suppression of the LRP16 gene expression inhibited MCF-7 cell growth and sensitized tumor cell to radiation [14-15]. Clinical data have shown that LRP16 is overexpressed in primary breast cancer samples compared with their matched normal tissues[16]. Recent, it was also reported that LRP16 is also an ERα coactivator[6]. Activation of ER signaling pathway plays important roles in multi-tissue development[11, 17-19]. ER signaling activation by LRP16 implies that LRP16 may display an important function in ERαtarget tissue development. To investigate the physiological function of the LRP16 gene, we constructed the LRP16 targeting vector and screened an ES cell clone with the target homologous recombination, which lays a foundation for raising LRP16 knockout mice.
Mouse LRP16 gene is 141 kb in length and composed of 11 exons and 10 introns. As concerned for gene targeting technology, the longer the homologisation sequence, the more probability of homologous recombination is. The exons between the fourth exon and the eleventh exon are relatively compact and are responsible for encoding a putatively conversed domain including within the LRP16 protein, so this region is ideal for gene targeting. Among theses exons, the shortest is 31 bp and the longest is 123 bp. According to the analytic results of the enzyme patterns and the positions of exon, the fifth exon is selected for target site of insertion inactivation by SA-IRES-βgeo fragment, which is usually used as an expression cassette of inserting inactivation [20].
In the process of insetting SA-IRES-βgeo, although SalⅠsite does not exist within the target region, it is ingeniously introduced into the fifth exon by utilizing SacⅡsite and an unrelated DNA sequence, which is finally displaced by SA-IRES-βgeo cassette.

REFERENCES

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小鼠LRP16基因打靶载体的构建和
同源重组型胚胎干细胞筛选*○

伍志强1，韩为东1，赵亚力1，司艺玲1，母义明2，孟元光3，Masatoshi Nomura○4
解放军总医院, 1分子生物室,2内分泌科，3妇产科，北京市 100853; 4日本九州大学医学院第三内科,生物调控研究室, 日本福冈 8128582
伍志强，男，1982年生，广西壮族自治区桂林市人，汉族，解放军第一军医大学毕业，技师，主要从事肿瘤分子生物学的研究。
通讯作者：韩为东，解放军总医院分子生物室，北京市 100853
国家自然科学基金资助项目（30670809）*
摘要
背景：LRP16基因是一个雌激素反应基因，其表达水平与乳腺癌细胞增殖及侵袭密切相关。
目的：构建针对小鼠LRP16基因的打靶载体，转染胚胎干细胞(Embryonic Stem Cell)并筛选同源重组克隆。
设计：通过SA-RIES-βgeo插入小鼠LRP16基因组DNA构建插入失活型打靶载体。
单位：日本九州大学第三内科生物调控研究室与解放军总医院分子生物室。
材料：本实验所用主要材料由日本九州大学生物调控研究室提供。实验所用鼠基因组文库（mouse genomic library in pBeloBAC11 Vector）购自invitrogen公司，TopF10感化态细菌购自北京天为时代公司，pCDNA3.1(+)由本室保存。鼠胚胎干细胞株由日本九州大学提供。
方法：实验主要工作于2004-11/2005-05在日本九州大学生物调控研究室与解放军总医院分子生物室完成。PCR方法筛选129品系小鼠基因组文库中LRP16克隆，在第5外显子内插入SA-RIES-βgeo序列，构建插入失活型打靶载体并转染胚胎干细胞，经G418筛选，挑取抗性克隆，Southern blot方法鉴定同源重组胚胎干细胞克隆。
主要观察指标：具有同源重组的胚胎干细胞克隆。
结果：将包含第5至11外显子的LRP16基因组片段亚克隆到pBluescript SK II +载体, SA-IRES-βgeo序列的正确插入第五外显子中，打靶载体成功转染胚胎干细胞，Southern blot结果显示具有一个打靶序列同源重组型插入的胚胎干细胞克隆。
结论：成功构建了第五外显子插入失活型LRP16基因打靶载体并筛选到同源重组型胚胎干细胞。
关键词: LRP16；基因打靶；胚胎干细胞
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)12-02391-05
伍志强，韩为东，赵亚力，司艺玲，母义明，孟元光，Masatoshi Nomura.小鼠LRP16基因打靶载体的构建和同源重组型胚胎干细胞筛选[J].中国组织工程研究与临床康复，2008，12(12):2391-2395
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2391(ps).pdf]
(Edited by Zhou SL/Su LL/Wang L)