The human COL1A1/COL2A1 chimeric gene in transgenic mice: Analysis of themRNA expression and the apparent inhibition of translation from the construct
Abstract
Studies examining tissue-specific elements of the COL1A1 promoter indicate that sequences from $-2.3$ to $-3.5$ kb are required for appropriate expression in transgenic mice. However, we recently reported that only 476 bp of promoter in a $5\sp\prime$ 1.9 kb COL1A1 fragment conferred tissue-specific expression of a promoterless COL2A1 gene in transgenic mice (Sokolov et al., JBC 270:9622, 1995). Using this COL1A1/COL2A1 chimeric gene, four independent mouse lines were generated. Here, we report that all four lines express mRNA from the COL1A1/COL2A1 construct in tissues of transgenic mice that normally express COL1A1. In the highest expressing line, the level of COL1A1/COL2A1 mRNA was 35-45% of the endogenously expressed mouse COL1A1. In situ hybridization confirmed that mRNA expression of the COL1A1/COL2A1 construct colocalized with that of the endogenous COL1A1. Western blot assays detected processed $\alpha 1$(II) chains, showing that mRNA from COL1A1/COL2A1 was translated. However, immunohistochemical staining for type II collagen was negative. Analysis of collagens from mouse skin by ion-exchange chromatography and cyanogen bromide cleavage indicated that less than 2% of collagens in skin were comprised of type II collagen. RT-PCR and sequencing analysis revealed no mutations preventing translation of the mRNA for COL1A1/COL2A1. Western blot analysis and electron microscopy showed secretion of $\alpha 1$(II) chains from the cells, with no intracellular accumulation of type II procollagen. These results indicated that translation of the COL1A1/COL2A1 mRNA was inhibited. Northern analysis revealed endogenous antisense messages in both cultured mouse cells and tissues. Antisense RNA to type II collagen was detected in control mouse tissues that normally synthesize type I collagen. No antisense RNA was detected in tissues that synthesize type II collagen. Antisense RNA was also detected by RT-PCR. These results confirm previous indications that 476 bp of promoter in a 1.9 kb COL1A1 fragment were sufficient to drive inappropriate expression of the COL2A1 gene in cells of transgenic mice that normally express COL1A1. Also, the results demonstrate that the translation of mRNA from the COL1A1/COL2A1 construct was specifically inhibited. Endogenous antisense RNA to type II procollagen may play a role in this unidentified mechanism of post-transcriptional down-regulation.
Subject Area
Biochemistry|Molecular biology|Cellular biology
Recommended Citation
Yuan, Constance Miao-Leng, "The human COL1A1/COL2A1 chimeric gene in transgenic mice: Analysis of themRNA expression and the apparent inhibition of translation from the construct" (1997). ProQuest ETD Collection - Thomas Jefferson University. AAI9727337.
https://jdc.jefferson.edu/dissertations/AAI9727337
