Construction And Identification Of PRL-3 And PRL-3(+)/APC(min/+) Transgenic Mouse Models

BACKGROUND & OBJECTIVEColorectal cancer is a common malignant tumor in China. The incidence of colorectal cancer ranks the fourth highest number on the cancer spectrum. In the world, the incidence of colorectal cancer ranks the third highest number in malignant tumors, and there is a trend to rise. The major cause of treatment failure and death of colorectal cancer patients is liver and lymph node metastasis. Micrometastases can be found in most of patients before radical operation, which is the direct cause of recurrence and metastasis of colorectal cancer. So it is necessary to explore the related factors of colorectal cancer metastasis, find early specific molecular marker for early diagnosis, screen key factors in the development of tumor and clarify the mechanism of metastasis. The current study focus of the colorectal cancer is on exploring an ideal drug target for clinical treatment.PRL-3, also known as PTP4A3, is a newly identified gene associated with tumor metastasis. It can encode a class of tyrosine phosphatase of small molecular weight. It is one of a small number of specific elements which are metastasis-related to colorectal cancer. In 2001, Saha published an article about the relation of PRL-3 and colorectal cancer metastasis in the "Science", which is the first report about the relation of PRL-3 and human malignant tumor. PRL-3 is an enzyme which expresses highly in the cells of liver metastases. It is an ideal target for drug therapy. Currently, a large number of experiments in vitro and in vivo showed that high expression of PRL-3 could promote cell growth, invasion, migration, malignant transformation and cancer metastasis. Moreover, PRL-3 plays an important role in tumor angiogenesis. However, it remains unclear about how PRL-3 act in the specific mechanisms and signaling pathways of tumor metastasis.Previous studies of our research team have showed that the expression of PRL-3 is closely related to metastasis of colorectal cancer. We found a new protein CDH22 (cadherin family member) which can interact with PRL-3 by yeast two-hybrid technology. Besides, we studied the regulatory mechanism of PRL-3 in colorectal cancer-related cadherin signaling pathway. However, tumor metastasis is a complex, multi-step and multiple genes involved process which can be regulated by many factors in human body. Therefore, in order to further clarify specific mechanism of PRL-3 in tumor metastasis and determine the role of PRL-3 gene in colorectal cancer metastasis of the general body, we construct PRL-3 transgenic animal model for annotating PRL-3 in vivo.We constructed a PRL-3 transgenic mouse model by pronuclear DNA microinjection. Meanwhile, we established PRL-3(+)/APC(min/+) transgenic mice by cross breeding PRL-3 transgenic mice with APC(min/+) mice, the latter is a heterozygous with APC mutation and spontaneous intestinal adenomas. Two transgenic mouse models were identified by biological molecular assay and inspected phenotype changes on individual development. Both of the mouse models can not only be used to study PRL-3 function in vivo, but also provide drug-target animal models for anti-metastatic chemotherapy in vivo. For ultimate destination, we hope that PRL-3(+)/APC(min/+) mice can develop into a spontaneous metastasis transgenic mouse model in accordance with the malignant transformation and progression of human colorectal cancer in the future.METHODS1. Construction and identification of mA33 intestinal specific promoter-driven human PRL-3 vectorThe intestinal specific expression mouse A33 promoter (mA33) is connected seamlessly with human PRL-3 gene full cDNA (removed stop codon TAG) by overlap PCR. The eukaryotic expression vector contains pEGFP-N1-mA33-PRL-3 is constructed by gene vector cloning technology, and then the vector clone is confirmed with restriction endonuclease analysis, PCR amplification and DNA sequencing.2. Establishment of PRL-3 transgenic mice model by pronuclear DNA microinjectionpEGFP-N1-mA33-PRL-3 (2.4 kb) is dissected from vector with restriction endonuclease. Purified 2.4 kb fragment is recycled from agrose gel, which is diluted as 2ng/μl DNA solution for microinjection. Fertilized eggs were donored by female and male mice. The recombinated DNA fragmants was injected into single-cell stage fertilized eggs with pronuclear DNA microinjection under microscopic manipulation. Pseudopregnant female mice were established from ligated male mice and female mice mating prior to transplantation of including pEGFP-N1-mA33-PRL-3 fertilized eggs. The treated eggs were transplanted into the oviduct of pseudopregnant female mice to develop F0 transgenic mice. Positive transgenic founders were verified preliminarily with repeated twice PCR. 3. Reproduction and identification of PRL-3 transgenic micePCR-positived transgenic mice mated with wild-type mice C57BL/6J. The offsprings of the founders were selected preliminarily through whole body fluorescence imaging and two-primer PCR detection. Then the offsprings were euthanatized at different month-old and identified by biopsy, histopathological examination, immunohistochemistry, fluorescence microscope EGFP detection and western blot. The integration of PRL-3 DNA, the expression of PRL-3 protein and EGFP production of transgenic mice were all analyzed in different months old offsprings.4. Genetic stability and phenotype analysis of PRL-3 transgenic mouse modelAfter obtaining the reproductive efficiency of positive transgenic mice, it is indispensable to analyze the genetic stability of PRL-3 transgenic mice. We also took records in detail of the positive transgenic mice offsprings, including their body shape, behaviour habits, individual development, hair, body weight and reproductive ability. Systemic pathological examination was performed for lesions combining with the phenotype of the offsprings.5. Breeding and identification of APC(min/+) miceAPC(min/+) mice were selected in the offspring which came from the mate breeding of APC(min/+) positive male mice and wild-type negative female mice by PCR. The positive mice were observed closely. APC(min/+) mutant mice and wild-type mice were euthanatized by stages (1m,3m,4m). N=6, male and female in half. The growing development and body weight of mutant mice and wild-type mice were compared. The formation and development of gastrointestinal adenomas were observed by general and biopsy pathological detection. The size (longest diameter) and the number of adenomas were measured by Image-Pro Plus (IPP) 5.0 image analysis software.6. Establishment and identification of PRL-3(+)/APC(min/+) transgenic mouse modelPRL-3 (+)/APC(min/+) transgenic mice were selected in the offspring which came from the mate breeding of PRL-3 transgenic mice and APC(min/+) transgenic mice by PCR. The positive mice were observed closely. PRL-3(+)、APC(min/+)、PRL-3(+)/APC(min/+) and wild-type mice (1m,3m) were sacrificed respectively, N =6, male and female in half. The individual development and body weight of the four one-month-old mice were compared. The formation and development of gastrointestinal adenomas were observed by general and biopsy pathological detection. The size (longest diameter) and the number of adenomas were measured by Image-Pro Plus (IPP) 5.0 image analysis software.7. Statistical methodsExperimental datas were analyzed by statistical package SPSS13.0. The weight of mice was analyzed by factorial designed variance analysis, One-way ANOVA and two independent samples T-test. The size of the intestinal adenomas was analyzed by two independent samples T-test.RESULTS1. PRL-3 vector drived by intestinal specific promoter mA33 was constructed successfully.EGFP-N1-mA33-PRL-3 vector was constructed successfully by overlapping PCR and cloning techniques. The vector was identified by enzyme digestion, PCR and DNA sequencing. The ultimate pronuclear DNA microinjection fragment was as follows:transcriptional protection sequence-promoter mA33-PRL-3 gene-green fluorescent protein EGFP-transcription terminator SV40 polyA. The total size of the fragment was 2.4 kb. Promoter mA33 and target gene PRL-3 were connected seamlessly. Then the PRL-3 gene and green fluorescent protein EGFP formed fusion protein by restriction sites connection. The whole injection fragment was designed properly and connected correctly.2. PRL-3 transgenic founders was generated successfully47 mice were obtained by the routine method of pronucleus-injection of fertilized eggs. Five positive transgenic mice expressing PRL-3 were identified by twice PCR detection. Transgenic positive ratio was 10.64%.3. PRL-3 transgenic mouse model was established successfullyWe derived two stable transgenic mouse lines from the five founders and have obtained F3 generation mice. The transgenic mice expressed both green fluorescent protein and PRL-3 were obtained too. Exogenous gene was integrated into DNA of transgenic mice that was identified by two-primer PCR. The localization of EGFP was consistent with PRL-3 protein in PRL-3 transgenic mice that was verified by immunohistochemistry and confocal inspection. Western-blotting results showed that EGFP and PRL-3 formed fusion protein, this indicated that visual PRL-3 transgenic mouse model was obtained. The main positive expressing regions are the brain, intestine, heart, lung, kidney, pancreas, et al. The transgenic mice with PRL-3 visualizing expression can not only act as an ideal animal model for PRL-3 function research in vivo, but also can be used for the preparation and screening of antitumor drugs in future. 4. Phenotype and genetic stability analysis of PRL-3 transgenic mouse modelThe results of DNA PCR tests have showed that the proportion of offspring carrying the exogenous was closed to 50%, which was consistent with Mendel’s laws. Transgenic mice acquired stable inheritance. By dynamic observation of 4 generations of PRL-3 transgenic mice we found some phenomenons:The proportion of positive male mice was significantly higher than female. There is no significant difference between male mice and wild mice in appearance, growth rate (P>0.05) and productivity. But female mice showed growth retardation (P<0.05), gentle temperament, sloth, inherently lower reproductive ability and decreased genetic ability. These results indicated that PRL-3 gene had obvious effect on growth, development and reproduction of the female mice. While the male mice acquired stable inheritance.Intestine specific promoter did not play an expected function in PRL-3 transgenic mouse model. But the expression level of PRL-3 was enhanced widely and that promoted the proliferation of lymph node lymphocytes of intestine and abdominal and the formation of new blood vessels significantly in these transgenic mice. One of the founders (38♀) showed a mass on its neck and has been survived, we will continue to monitor its tumor evolution and make sure the source of tumor until it is moribund.5. The growth-phenotype and spontaneous intestinal adenomas of APC(min/+) miceBy dynamic observation, we found that APC(min/+) mice grew slowly compared with wild-type mice (P<0.05). Bloody stool can be found in APC(min/+) mice of 2-month old. On 3-month old, most of APC(min/+) mice suffer from prolapse, persistent bleeding and ascites, and they were moribund at 4 month. They also showed some symptoms, such as anergy, bradycardia, hypopnea and anemia. The body weight of 4-month-old APC(min/+) mice were lighter than that of wild-type mice (P<0.05). The survival time was about 120 days.Spontaneous intestinal adenomas showed progressively increasing number of tumors and tumor volume in APC(min/+) mice. The adenomas in small intestine occured within the first month, tumor number was 20 to 50 and the tumor diameter was 0.6-5.5mm. While developed in large intestine more than one month, tumor number was 1-5 and the tumor diameter was 1.2-4.2mm. This showed adenomas in small intestine of APC(min/+) mice were more prominent.6. Establishment and identification of PRL-3(+)/APC(min/+) transgenic mouse modelWe obtained PRL-3、APC(min/+)、PRL-3(+)/APC(min/+) and wild-type mice by crossing PRL-3 transgenic mice with APC(min/+) mice. The body weight of PRL-3, APC(min/+), PRL-3(+)/APC(min/+) and wild-type mice were compared. The PRL-3, APC(min/+) and PRL-3(+)/APC(min/+) mice grew more slowly than the wild-type mice(P<0.05); While there was no significant difference among PRL-3, APC(min/+) and PRL-3(+)/APC(min/+) mice(P>0.05)PRL-3(+)/APC(min/+) mice had some special appearances and characteristicses, such as rough skin, sparse hair and growth retardation. The numbers of adenomas in the small intestine or large intestine of the PRL-3(+)/APC(min/+) mice were larger than APC(min/+) mice’s (P<0.05).In addition, a large number of obvious atypical mini-adenomas had be found in the colon of the former by histopathologic examination. CONCLUSIONS1. The recombinant plasmid pEGFP-N1-mA33-PRL-3 was constructed successfully. This plasmid provides a high quality three-gene integration vector for the study of colorectal cancer and PRL-3 function.2. Visualization of PRL-3 in transgenic mouse model with stable inheritance and expression was established successfully. It can provide an ideal animal model for the research of function and mechanism of PRL-3 in vivo.3. The enhanced expression of PRL-3 plays an important role in the hyperplasia of blood vessels and lymph nodes.4. PRL-3 can affect female mice in the growth, development and reproduction. The positive rate of male mice was significantly higher than female mice.5. PRL-3(+)/APC(min/+) mice were obtained by crossing PRL-3 transgenic mice with APC(min/+) mice. PRL-3(+)/APC(min/+) mice had some phenotypic changes, such as rough skin, sparse hair and growth retardation. Histopathologic examination showed a large number of intestinal adenomas and colorectal focal mini-adenomas in PRL-3(+)/APC(min/+) mouse model, the latter appeared obvious atypia and malignant trend.PROSPECT1. PRL-3 transgenic mouse model with wide enhanced and visualizing PRL-3 expression can be detected continuously or dynamically without damage in vivo. It will be an ideal animal model for cancer gene therapy. 2. PRL-3 transgenic founder 38♀has been found neck tumor. Whether we will obtain a stable germline with spontaneous tumor from PRL-3 transgenic mouse model through long-term selective breeding?3. At present, PRL-3(+)/APC(min/+) mice containing APC gene mutation and PRL-3 gene overexpression have been found mini-adenomas with obvious atypia and malignant trend in the large intestine. During a long-term cultivation, they are expected to develop into a spontaneous tumor metastasis transgenic mouse model which is consistent with the colorectal cancer patients in disease development.