Biochemistry and Molecular Biology Core

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Mouse breeding and characterization: An important function of Core B that will be utilized by all components involves the breeding and characterization of conditional LKB1 and SIRT1 mice. The "mouse" component of Aim B will implement a cost-effective facility for the production and maintenance of new mouse strains. The core will perform initial experiments and continually update methods associated with Cre-mediated transgene expression. The core will optimize the production of the mouse models and will provide training to investigators in the Ruderman, Cohen and Walsh laboratory regarding the optimal methods associated with the excision of SIRT1 and LKB1 transgenes. The core will provide investigators with mice to be amplified for their individual experiments. The core will construct conditional SIRT1- and LKB1-deficient mice predominantly using VEcad-Cre- ERT2 (1), but will also use Tie2-Cre if necessary (2). This core will utilize at least 2 transgenic strains of Cre recombinase to circumvent problems that could arise from potential embryonic lethality as well as from the potential lack of endothelial cell specificity of the Cre transgene. The major rationale for the Tie2-Cre transgenic line is that these mice can be obtained commercially as certifiably pathogen-free (The Jackson Laboratory), and work with this strain does not involve an induction protocol. Thus, we are able to initiate our experiments sooner and there is much prior experience with this line. However, we recognize that the Tie-2 line expresses Cre in approximately 90% of cells of the hematopoietic lineage (M. Luisa Iruela-Arispe, personal communication), despite some claims to the opposite in the literature. Thus, the core will undertake a second approach using the tamoxifen-inducible Cre-recombinase under the control of the VE cadherin promoter, and we have obtained these mice from Dr. Iruela-Arispe. This approach is potentially more powerful because the transgene can be induced at different stages of embryonic and postnatal development, it can give rise to a high frequency of endothelial-specific recombination in adult mice, and under some induction conditions can result in a minor (0.3%) subpopulation of bone marrow cells that are positive for the transgene (1). Construction ofSIRTI- and LKB1-deficient mice- The core will utilize the conditional Cre-loxP gene regulation system where the transgene expression is disrupted in a tissue-specific manner. LKB1-floxed mice (3) in the FVB/N background have been provided by Reuben Shaw (The Salk Institute). The SIRT1-floxed mice in a C57/BI6 background were provided by Drs. Sinclair and Alt. The breeding scheme is shown in the Figure. Genotyping will be performed by the core using PCR methods. The core will also backcross mice into the C57/BI6 background when required. The core will also provide investigators with control mice. In most cases control mice will be transgenic (Tg)-Cre-LKB1/SIRT1(+/+)mice. The core will characterize the degree of transgene excision. Using the tamoxifen-inducible Cre in adult mice, endothelial-specific recombination is reported to range from 71-92% (1). For adult experiments, tamoxifen (2 mg) can be delivered IP for 5 consecutive days. To delete LKB or SIRT1 in embryos, tamoxifen is injected IP into pregnant females at specific embryonic stages, and it is reported to achieve 95% excision frequencies (1). Of relevance for endothelial vs. hematopoietic excision, constitutive VE-cadherin-Cre displays a 50-60% penetrance in hematopoietic lineage cells (4). However, the VEcad-Cre-ERT2 can be induced with tamoxifen in adult mice and show a minimal (-0.3%) fraction of bone marrow cells that are positive for recombination (1). Thus, the VEcad-Cre-ERT2 model can potentially be very useful for studies proposed in the PPG because it can be used in a manner to promote excision of LKB1 or SIRT1 in the endothelium vs. hematopoietic lineage and it potentially circumvents problems associated with embryonic vascular malformations and lethality. It should be noted that the Walsh lab is familiar with the manipulation of strains of inducible transgenic mice (5-7), and Walsh lab personnel will materially participate in the operation of Core B which will provide my lab and the other PPG investigators with the "floxed" LKB1 and SIRT1 mice. >4na/yses of LKB1/SIRT1 excision- A number of analyses will be performed to assess the ablation of transgene in the endothelium. First, immunohistochemical analyses with LKB1 or SIRT1 staining will be performed on artery sections from wild-type and floxed mice. Second, LKB1 expression can be assessed in cultured endothelial cells isolated from wild-type, heterozygous- or homozygous-deficient mice by Western blot or real-time PCR analysis. Third, the core will estimate the degree of LKB1/SIRT1 excision by performing parallel experiments on mice where the Tg-Cre strain is crossed with ROSAR26R reporter mice that have a floxed 'stop' cassette upstream of LacZ. Thus the degree of excision can be assessed under different experimental conditions by analyzing histological sections from embryonic and adult mice. These experiments may be warranted because the degree of excision can be dependent on background strain, age, weight and pathological situations (1), and analysis with the ROSAR26R reporter mice would provide information about the behavior of the conditional Cre-loxP gene regulation system under the conditions of our assays. Biochemistry Component (A.K. Saha. PI) (Y. Ido CO-PI): This core will carry out selected analyses that will be utilized by 2 or more projects in the program. In addition, it will assist investigators in setting up certain assays that they will use intensively in their own laboratories. We anticipate that the measurements could include the following a) Tissue and endothelial cell concentrations of adenine nucleotides, (all projects), lactate, pyruvate, malonyl CoA, diacylglycerol, ceramides and triglycerides {S. Itani Diabetes 2003; Saha, AJP 1997} and NAD and NADH and plasma FFA levels. All of these measurements have been carried out by Dr. Saha in connection with studies of malonyl CoA and AMPK regulation in muscle and liver (DK19514), and/or by Dr. Ido in vascular cells (see Project 1 Literature Cited section for full references). It is anticipated that these measurements will be of especial use in determining the basis for AMPK activation (energy state vs redox), and in characterizing the tissue lipid abnormalities induced by the high fed diet when it stimulates atherogenesis. b) Assays related to SIRT1/LKB1 signaling. Dr. Lan working with Dr. Ido has set up assays for LKB Kinase (LKB tide) and many other parameters related to the SIRT1/LKB1 signaling mechanism (see project 1, Aim 1). They will be made available to other program investigators. c) Protein Kinase C: western blots and in some instances activity measurements of specific PKC isoforms (after immunoprecipitation) will be performed {Itani 2003}. Dr. Ido has set up most of these methods for endothelial cells and Dr. Saha has a long experience in studying PKC in other tissues {Itani, S., et. al., Diabetes 2003; Saha, A.K., et. al. Am. J. Physiol. 1997}. Based on recent experience, an increasing use of these methods by all projects is anticipated. 3. Molecular Biology and Cell Culture Components (Y. Ido. PI: K. Walsh. Consultant). A. Viral Constructs and Cell Tagging: Dr. Lan in Dr. Ruderman's lab has been marking and cataloging a number of adenoviruses (listed later) including those for LKB1, LKB1 various mutants, SIRT1 and SIRT1 dominant negative mutants, and dominant negative and constitutively active AMPK. In addition to these adenoviruses, she has made lentivirus expressing shRNA for human and mouse SIRT1. Dr. Ido's lab has been adopting a system which upon request, allows him to produce many different tagged (including flag, his, GST, GFP, DsRed) and backbone (adenovirus, lentivirus, AAV, bacteria and insect expression) constructs using the Gateway system (Invitrogen). His lab has also produced NF-kappaB reporter adenoviruses that detect NF-kB activation status in endothelial cells. The core will provide services to produce these adenoviruses by amplification and purification, as well as lentivirus and various tagged constructs upon request. B. Antibodies: The second task of this core will be to provide information about commercially available and custom made antibodies. Dr. Lan has accumulated information concerning the antibodies relevant for all three projects including those for AMPK, p-AMPK and LKB1. The latter include antibodies for pS428 LKB1 and autophosphorylated (pT336) LKB1, and acetyl-lysine antibodies. The core will provide such information to individual laboratories. Our studies indicate that SIRT1 induced acetylation of LKB1 occurs at K48. Therefore, we will produce a specific antibody to detect LKB1 K48 acetylation. If the antibody is successfully made, the core will distribute it to the individual labs. Dr. Lan will carry out these activities under Dr. Ido's supervision. C. Maintenance of BAECs and HAECs: The third task of this core will be to culture bovine and human aortic endothelial cells (BAEC and HAEC) and distribute them to individual labs. The purpose is to avoid variations that might result from culturing cells from different sources in the individual labs. . In addition, to avoid variability amongst different human donors, the core will buy HAECs from several donors, pool them and provide the individual labs with "standardized" HAECs. Since these cells may change in character with higher passage number, the core will also periodically add younger passage cells to the pool and then re-characterize them based on various characteristics to assure relative biological constancy. D. To assist the Animal Core in the production of cultured endothelial cells from mice with endothelial cell-specific SIRT1 and LKB1 deletion mutations. Dr. Walsh's lab is experienced in culturing mouse lung endothelial cells, and these cells from from flox'd SIRT and LKB1 will be cultured and provided by the core so that individual projects may use them as a knockdown strategy