1.Construction Of The Sla/lp Gene Knock-out Mouse Model 2.Identification And Characterization Of The Sla/lp Gene Promoter

[Background] Autoimmune hepatitis (AIH) is one of the three putative autoimmune liver diseases afflicting human beings worldwide, but affects mainly wamen and children. Most of the AIH patients respond to immunosuppressive therapy quite well but have a poor prognosis if untreated (Krawitt 1996). The key is to give a correct diagnosis in the early stage of AIH. However, there is no specific laboratory hallmark for diagnosing AIH. The diagnosis of AIH is based on assessment of clinical and serum biochemical (elevated serum IgG) features, histologic abnormalities and the presence of autoantibodies, such as antinuclear antibody (ANA), smooth muscle antibody (SMA), anti-liver-kidney microsome-1 antibody (LKM) and perinuclear anti-neutrophilic cytoplasmic antibody (pANCA). The sole exception is anti-soluble liver antigen/liver-pancreas antigen antibody (anti-SLA/LP), which is the exclusively specific marker with 100% specificity and 20% sensitivity for AIH (Wies et al. 2000).SLA/LP protein is expressed widely in normal tissues as well as in embryo. SLA/LP is a cytosolic protein of about 50 kDa expressed in enzymatically active organs such as liver, pancreas, kidney, lung, testis and overexpressed in activated lymphocytes. It may be an enzyme or a group of enzymes (Wies et al. 2000). Another study found the SLA/LP molecule was associated with the UGA serine tRNA-protein complex (Costa et al. 2000), which facilitates the co-translational incorporation of selenocysteine into proteins, it has been speculated that the SLA/LP molecule may have a role in selenoprotein metabolism. The nature and the biological function of the SLA/LP molecule was reported recently. It is believed that the SLA/LP protein functions as phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which generates selenocysteinyl-tRNA^Sec from a phosphoseryl-tRNA precursor, and is a key enzyme catalyzing selenocysteine - the twenty-first amino acid to synthesis selenoprotein in eukaryotes and archaea (Chambers et al. 1986; Zinoni et al. 1986; Yuan et al. 2006; Xu et al. 2007). However, the regulation of its expression and the mechanism of autoimmunisation are not clear so far. It will be helpful for understanding autoimmunity to SLA/LP and the pathogenesis of AIH, if we knew more about SLA/LP.To address the function of SLA/LP protein, we are going to generate sla/lp gene knockout mice to study the changes of the phenotype. This mouse model will also be helpful to understand AIH well. Furthermore, we are going to map the promoter position and possible transcription factors, which will show us how the expression of SLA/LP protein is regulated and may give us hints about the pathogenic autoimmunty to SLA/LP in AIH.Part I: Construction of the sla/lp Gene Knock-out Mouse Model[Aim] To construct a sla/lp gene knockout mouse model, study the changes of its phenotype and offer an ideal mouse model for study AIH and the function of SLA/LP.[Methods] (1) A sla/lp gene knockout vector was constructed by several cloning processes. (2) The sla/lp knockout vector was transfected into ES cells by eletroparation in order to knockout part of sla/lp gene by homologous recombination. (3) The drug resistant ES clones were selected by G418 and ganciclovir, and the positive recombinant ES clones were confirmed by Southern blot. (4) The positive ES cells were microinjected into 3.5-day blastocysts from C57BL/6 mouse, transplanted to foster mother in order to get chimeras. (5) The male chimera was breeded with wildtype C57BL/6 female mice for getting heterozygous mice. (6) To breed heterozygous mice for getting homozygous gene knockout mice. (7) To analyse the phenotype of sla/lp gene knockout mouse.[Results] The constructed knockout vector contained two pieces of homologous sequences of sla/lp gene, named 5' Arm and 3' Arm. The 5' Arm was 2007bp from intron 2-3 to exon 3. The 3' Arm was 4256bp from intron 6-7 to exon 8. The knockout fragment therefore was 5867bp from exon 3 to intron 6-7 after homologous recombination. The EGFP gene was inserted into the knockout vector immediately after 5' Arm. There was a positive selection marker - neomycin gene located between the 5' Arm and 3' Arm, while a negative selection marker - HSV-tk gene was located outside of them. After electroparation, there were many drug resistant clones after ganciclovir and/or G418 selection. However, three positive ES clones were comfirmed after screening more than 400 clones by Southern blot.There were seven male chimeras born with high chimerism with brown-black coat. However, all the offsprings were black and neomycin negative after breeded with C57BL/6 wildtype female mice, which indicated there was no germline transmission. [Conclusions] The sla/lp gene knockout vector was constructed successfully and it could result the naissance of chimeras. However, there was no germline transmission. The problem might be overcome by repeating microinjection with another positive ES clone or constructing a new knockout vector.Part II: Identification and Characterization of the sla/lp GenePromoter[Aim] To identify the mouse sla/lp gene promoter and possible transcription factor binding sites to understand the regulation of the sla/lp gene expression in normal tissues, and may help to understand the pathogenic autoimmunty to SLA/LP in AIH.[Methods] (1) A 1740bp of the sla/lp gene fragment was amplified by PCR and subcloned into luciferase reporter vetor - pGL3 basic vector. (2) The constructed vector was transformed into three cell lines, HEK293, Hepa1-6 and RAW264.7 for luciferase assays. (3) After testing the provocation function of the 1740bp fragment, a series of 5'-end or 3'-end deletion clones were constructed to localize the sla/lp gene core promoter position. (4) The possible transcription factor binding sites were predicted by computer program and tested with point mutant clones by luciferase assays. (5) The possibility of each binding site was further confirmed by gel shift assay and super gel shift assay.[Results] (1) The luciferase assay showed that the 1740bp sla/lp promoter fragment could initiate the transcription of luciferase and was orientation dependent. (2) The basic fragment initiating transcription was 63bp located at -99 to -37. (3) There were no typical core promoter elements within the 63bp fragment, such as TATA box, Inr, DPE and BRE. (4) According to CpGProD and AliBaba2.1, the mouse sla/lp promoter was a CpG island promoter and there were two Sp1 binding sites (-85 to -76 and -55 to -41) and one RAP1 binding site (-71 to -62) were predicted within the 63bp mutant. Furthermore, an Oct-1 binding site (-184 to -175) was predicted upstream of this fragment. (5) The luciferase activity was vanished by point mutant clone of Sp1 or RAP1 binding site, while, the mutant clone of Oct-1 binding site could increase the luciferase activity by 2.8 to 6.3-fold in different cell lines. (6) The gel shift assays of the four predicted transcription factor-binding sites gave a shifted band seperately. However, only the Sp1 binding site at -85 to -76 or the Oct-1 binding site could result a super shift. The results indicated the existance of Sp1 (-85 to -76) or Oct-1 binding site, while the Sp1 binding site at -55 to -41 or the RAP1 binding site could not be confirmed.[Conclusions] (1) The core promoter of the mouse sla/lp gene was localized within a 63bp fragment located at -99 to -37 upstream of transcription initiation site. There was no typical core promoter element inside the core promoter. There was a Sp1 binding site (-85 to -76), a Sp1-like binding site (-55 to -41) and a probable RAP1 binding site (-71 to -62) in this fragment, which functioned as transcription activators. There was also a repressor, Oct-1 binding site (-184 to -176), located upstream of this core promoter. (2) The expression of SLA/LP protein seems to be the result of a balance between activator Sp1/RAP1 and repressor Oct-1. (3) The factors causing unbalance of the function between activator Sp1/RAP1 and repressor Oct-1 may be one of the reasons resulting anti-SLA/LP antibidies in AIH patients.[Summary] The effort to construct a knockout mouse model was fail in the first try because there was no germline transmission even though the chimeras were highly chimerism. Therefore, the mutant gene could not be transferred to the offsprings. This might be caused by many reasons. The key point to solve this problem is to avoid the differentiation of ES cells. The way to solve the problem is to repeat microinjection with another positive ES clone or re-construct a knockout vector, which is being carried on.In this study, we focused mainly on the transcriptional regulation of the murine sla/lp gene. We mapped the core promoter region of the sla/lp gene, which is localized within a range from -99 to -37 flanking the 5'-end of transcription start site. Within this core promoter area, a Sp1 binding site, a Sp1-like binding site and a possible RAP1 binding site were identified. All of these cis-activating elements are essential for maintaining the basic initiation of transcription of sla/lp gene. 75bp upstream of this core promoter, there is an Oct- 1 binding site that acts as a repressor down-regulating the expression of SLA/LP protein.The sla/lp gene promoter is a CpG island promoter, which normally contains multiple transcription start sites. This promoter lacks typical core promoter elements, such as TATA box, Inr, DPE or BRE. The structure of the sla/lp promoter is a multiple regulator architecture indicating the sla/lp gene is regulated in a complicated combinational way.The expression of the SLA/LP protein seems to be the result of a balance between activator Sp1/RAP1 and repressor Oct-1. The factors causing function unbalance between activator Sp1/RAP1 and repressor Oct-1 may be one of the reasons resulting anti-SLA/LP antibidies in AIH patients. It would be very interesting to find out these factors.All the transcription factors identified, Sp1, RAP1 or Oct-1, are expressed ubiquitously. The non-tissue specific expression of SLA/LP protein may be due to the non-tissue specific transcription factors. The concentration and activity of these factors as well as some liver/pancreas specific signals may contribute to the transcription frequency of the sla/lp gene in different tissues. Given the almost ubiquitous expression of SLA/LP, the question then is, why pathogenic autoimmunity to SLA/LP only affects the liver and not other organs. A possible explanation was that aberrant expression levels of SLA/LP in liver might drive SLA/LP autoimmunity. However, our another result could demonstrate that the hepatic expression levels of sla/lp in patients suffering from autoimmune hepatitis was not distinct from that of healthy subjects or patients with other liver diseases (Wang et al. paper unpublished). An alternative explanation for the liver-specific pathology despite nearly ubiquitous SLA/LP expression could be that some modification of SLA/LP molecules in the liver may drive autoimmunity. Indeed, nutritional selenium is metabolized mainly by the liver, from where it is distributed to other organs in the form of selenoprotein P20 (Gromer et al. 2005; Schweizer et al 2005). Thus, it is possible that nutritional selenium may form neoantigenic ribonucleoprotein complexes, which then may drive autoimmunity to SLA/LP by similar ways to those found in immune-mediated drug-induced hepatitis (Liu et al. 2002). However, further research is needed to clarify this issue.The expression pattern and the promoter features suggest strongly that the mouse sla/lp gene may be a housekeeping gene.