Analysis of Tissue-Specific Expression of Human Type II Collagen cDNA Driven by Different Sizes of the Upstream Region of the β-Casein Promoter

Abstract

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning

“…Naruse and collaborators used 1.8-kb and 3.1-kb fragments of the β-casein promoter with the untranslated first exon of the β-casein gene and the poly-A signal of bovine growth hormone. They observed low expression from the 1.8-kb promoter and suggested the 3.1-kb promoter can be used to induce mammary gland-specific expression of transgenes in transgenic animals (Naruse et al, 2006). Other authors (Sohn et al, 1999(Sohn et al, , 2003 have used a 10-kb fragment including untranslated exon 1, intron 1, and the 5'-UTR of exon 2.…”

“…This result suggests that the Pβcas5 fragment more efficiently expresses recombinant proteins, and its use for the production of transgenic animals may be preferred over the previously suggested 3.1-kb (Naruse et al, 2006) and 3.8-kb (Cerdan et al, 1998) promoters. Expression of hFIX was verified by whole mRNA from the cDNA of transgenic BMECs by PCR ( Figure 2B).…”

“…Human growth hormone expression in milk was driven by a 3.8-kb promoter of mice (Cerdan et al, 1998). Human type II collagen was expressed from 1.8-and 3.1-kb bovine β-casein promoters in transgenic mice (Naruse et al, 2006). A 3.7-kb promoter fragment was used to express human follicle-stimulating hormone in transgenic mice (Kim et al, 2007).…”

“…The reporter gene chloramphenicol acetyl transferase was expressed in transgenic bovine mammary epithelial cells (BMECs) from a 950-bp promoter fragment (Ahn et al, 1995). Others have proposed that 3.1- (Naruse et al, 2006) and 3.8-kb (Cerdan et al, 1998) promoters are sufficient for the production of transgenic animals.…”

“…In our constructs, the hFIX cDNA sequence was used without introns, which may reduce mRNA stability in BMEC culture. Other groups have used a poly-A signal from growth hormone (Naruse et al, 2006) or the cloned gene (Schnieke et al, 1997). We used the poly-A signal located in the "R" region of the 3'-LTR of the lentiviral vector.…”