Analysis of the promoter region of human cartilage oligomeric matrix protein (COMP).

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein expressed in cartilage, ligament, and tendon. The importance of COMP in the matrix of these cells is underscored by the discovery that mutations in COMP cause the skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Here, we present the first report on the analysis of the human COMP promoter region in cartilage, ligament, and tendon cells. A 1.7-kb region of the COMP promoter has been cloned and sequenced and no TATA or CAAT boxes were found. Primer extension identified multiple transcription start sites. All four transcription start sites were utilized in chondrocytes with only three of them utilized in tendon and ligament cells. Differential regulation was observed for different parts of this 1.7-kb region with the 370-bp proximal region conveying the strongest promoter activity. The highest activity was observed in tendon and ligament. Finally, we provide evidence that the DNA binding protein SP1 plays a role in the regulation of COMP expression. These results indicate that COMP expression within these cells is regulated in a unique manner that differs from the expression of other extracellular matrix genes.

Analysis of possible WT1 RNA processing in primary Wilms tumors.

WT1 RNA processing abnormalities have been suggested to play a role in the development of Wilms tumor by reports of editing at codon 280 in the rat WT1 transcript (codon 281 in humans) and aberrant splicing of exon 2 in WT1 transcripts from Wilms tumor xenograft cell lines. Both events result in a functionally changed WT1 protein and are potential mechanisms of altering normal protein function in the absence of WT1 DNA mutations. To determine whether either of these RNA processing events occurs in primary Wilms tumors, we analysed WT1 mRNA from 15 primary tumors. There was no evidence of WT1 RNA editing at codon 281, and only one primary tumor displayed aberrant splicing of exon 2. Sequence and Southern analysis of DNA from this tumor did not reveal any alteration in or around exon 2. These results suggest that neither RNA editing at codon 281 nor aberrant exon 2 splicing is a frequent mechanism of WT1 alteration during tumorigenesis.

Retroviral delivery of DNA into the livers of transgenic mice bearing premalignant and malignant hepatocellular carcinomas.

To develop gene therapy for hepatocellular carcinoma (HCC), we infused mice through the portal vein with retrovirus carrying the Escherichia coli beta-galactosidase reporter gene under the transcriptional control of the viral long terminal repeat (LTR) and the promoter from the mouse multidrug resistance gene mdr1b. Two transgenic mouse HCC models were used, one bearing the human hepatitis B viral envelope protein and the other SV40 T antigen. These animals develop HCC with predictable pathological manifestations. The viral transduction efficiency appeared to depend upon the stage of the disease in the animals. The most efficient transduction occurred when the livers had developed microscopic nodular hyperplasia; in some cases as many as 0.01-0.1 copies/cell were transduced. The transduction efficiency was lower in the late stage of the disease when livers had a heavy tumor burden and in the early stage when no lesion was evident. Low viral transduction efficacy was also seen in nontransgenic animals but was significantly increased by partial hepatectomy. The expression of the reporter gene in these animals was very low, as determined by histological staining. These results suggest that hepatocarcinogenesis can enhance retroviral delivery of foreign genes into the liver. Further development by increasing the viral transducing efficiency and the level of expression of transduced gene is required.

FVB/N: an inbred mouse strain preferable for transgenic analyses.

FVB/N mice offer a system suitable for most transgenic experiments and subsequent genetic analyses. The inbred FVB/N strain is characterized by vigorous reproductive performance and consistently large litters. Moreover, fertilized FVB/N eggs contain large and prominent pronuclei, which facilitate microinjection of DNA. The phenotype of large pronuclei in the zygote is a dominant trait associated with the FVB/N oocyte but not the FVB/N sperm. In experiments to generate transgenic mice, the same DNA constructs were injected into three different types of zygotes: FVB/N, C57BL/6J, and (C57BL/6J x SJL/J)F1. FVB/N zygotes survived well after injection, and transgenic animals were obtained with efficiencies similar to the F1 zygotes and much better than the C57BL/6J zygotes. Genetic markers of the FVB/N strain have been analyzed for 44 loci that cover 15 chromosomes and were compared with those of commonly used inbred strains. In addition to the albino FVB/N strain, pigmented congenic strains of FVB/N are being constructed. These features make the FVB/N strain advantageous to use for research with transgenic mice.