Conditional Expression Of Human Cripto-1 In Transgenic Mice Mediated By Cre/lox P System

BACKGROUND & OBJECTIVEHuman Cripto-1(CR-1), also known as teratocarcinoma-derived growth factor-1 (TDGF-1), is a member of the epidermal growth factor-cripto FRL1 cryptic (EGF-CFC) family, which is indispensable for early embryonic development. CR-1, which is identified as a marker for embryonic stem cells and generally absent from adult tissues, was overexpressed in human breast, colon, lung, testicular, stomach, pancreatic, and ovarian cancers. In transgenic mice, overexpression of human CR-1 under the control of breast-specific promoter initated the oncogenesis in mouse breast, suggesting that CR-1 plays an oncogenic role in breast cancer.Usually, the best and typical method to confirm one gene serving as an oncogene is that target gene is overexpressed in transgenic mice, following by observing pathological changes. The CR-1 transgenic mice(mentioned above), in which CR-1 gene under the control of breast-specific promoter is overexpressed in breast, could not be employed to examine whether CR-1 overexpression could also give rise to cancers in other organs of mice.In order to simulate the process of oncogenesis induced by potential oncogene (i.e., human CR-1) in vivo, The transgenic mice with the conditional expression of human CR-1 mediated by Cre/lox P system are required to be developed, which provides us with an ideal disease model for cancer pathogenesis. METHODS1. Construction of pRCLGHuman CR-1 cDNA was amplified from pCI-cripto-1 by PCR, and then inserted into the Sma I site of pRLG by In-Fusion PCR Cloning System to create pRCLG, as verified by sequencing and restriction enzyme digestion.2. Generation of RCLG transgenic miceRCLG transgenic mice were generated by microinjection of single-cell embryos using standard techniques as previously described(Nagy et al.,2003). The FVB mouse strain was used as the source of embryos for the micromanipulation and for subsequent breeding trials. All transgenic lines were created on the FVB genetic background. For microinjection, pRCLG was firstly cut by SfiⅠ, and subsequently via SspⅠto release the-9.436kb fragment of transgene RCLG. The injected fragments of RCLG were isolated and purified using the QIA quick gel extraction kit (Qiagen, Hilden, Germany), diluted to a final concentration of 2μg/ml DNA injection buffer(10 mM Tris/0.1 mM EDTA, pH 7.4), and microinjected into the pronuclear of one cell-stage fertilized embryos [FVB mouse(♀)×FVB mouse(♂)]. Then 20-25 DNA-injected DNA fertilized eggs that survived microinjection were implanted into the oviducts of one recipient pseudopregnant ICR mouse (Nagy et al.,2003) 2-3h after injection or next day as described (Nagy et al.,2003), and developed to term, followed by preliminarily screening mRFP transgenic mice from potential transgenic founders via mRFP assay under stereo fluorescence microscope and/or by using small animal in vivo imaging system(Xenogen, IVIS Lumina-Ⅱ) at birth & 3 weeks after birth, and subsequently confirming the results of mRFP assay by PCR-based genotyping (3 weeks after birth) to screen genomic DNA from mouse tail biopsies prepared with standard protocols(Sambrook et al,2001) for the presence of RCLG transgene (data not shown).3. Mouse propagation and transmissionAt 6-8 wk of age, founder shown to be transgenic for mRFP was mated with wildtype FVB mouse to produce F1 generation, followed by mRFP assay under stereo fluorescence microscope and/or by using small animal in vivo imaging system (Xenogen, IVIS Lumina-Ⅱ) to demonstrate whether mRFP transgene in RCLG transgenic mice (F0) could be transmitted to the next generation (F1).4. Detection of mRFP expression in adult organs of RCLG transgenic mice under stereo fluorescent microscope and by using small animal in vivo imaging system5. Conditional expression of human CR-1 in RCLG transgenic mice mediated by Cre/lox P systemRCLG transgenic mice were mated with the different Cre transgenic mouse lines (Ella-Cre transgenic mice and Alb-Cre transgenic mice) to produce double transgenic mice, in which Cre mediated the DNA recombination to activate the Luc and CR-1 expression which could be detected by in vivo bioluminescence imaging and several molecular methods, such as RT-PCR and Western blot.RESULTS1. Generation of RCLG transgenic miceTransgenic construction of pRCLG was constructed by standard molecular approaches, following by confirming that the newly-constructed pRCLG was correct by sequencing and enzyme digestion. Two mice (190# and 225#) exhibited red fluorescence assayed by small animal in vivo imaging system and under stereo fluorescence microscope, as confirmed by PCR analysis for the RCLG transgene integrated into the genome, indicating that two founders were successfully attained. In addition, F1 and F2 progeny inherited and expressed mRFP transgene from RCLG transgenic founder (F0).2. mRFP expression in adult organs of RCLG transgenic mice (F2)Under stereo fluorescence microscope, red fluorescence could be detected in different adult organs including testis, kidney, stomach, intestine, lung, liver, brain, thymus, spleen and heart. Furthermore, the heart, testis, brain, stomach, kidney and intestine showed strong red fluorescence, while the lung, liver, thymus and spleen showed relatively weaker signals.3. Conditional expression of human CR-1 in RCLG transgenic mice mediated by Cre/lox P systemBy in vivo bioluminescence imaging, Luc expression activation was detected in liver of double transgenic mouse(RCLG/Alb-Cre mouse), but not in all organs of double transgenic mouse(RCLG/EⅡa-Cre mouse).CONCLUSIONS1. Two RCLG transgenic founders were successfully generated, and RCLG transgene could be transmitted from founders to subsequent generation.2. Normal expression of mRFP transgene in multi-organs of RCLG transgenic mice.3. Conditional expression of human CR-1 in the liver of RCLG transgenic mice mediated by Cre/lox P system was realized.