A 3,387 bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

Abstract

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.

Production of recombinant human proinsulin in the milk of transgenic mice

“…We used a qPCR technique to estimate the copy number of the transgene in each line of the transgenic mice16. The results indicated that the transgene copy numbers in the founders were different, ranging from 7 to 20.…”

“…The transgene copy number in the transgenic mice was determined by qPCR as described previously16 with primers designed for a single copy control gene (lymphotoxin B gene: Ltb) and the pBC1-INS transgene. The primer sequences for Ltb have been described previously16, and the primer sequences for the pBC1-INS were pBC1-INS-F and pBC1-INS-R (Table 3).…”

“…The primer sequences for Ltb have been described previously16, and the primer sequences for the pBC1-INS were pBC1-INS-F and pBC1-INS-R (Table 3). The PCR products for Ltb were linked to those of pBC1-INS, and the resulting Ltb-pBC1-INS DNA fragment was cloned into the pCR-Blunt II-TOPO vector (Invitrogen) and transformed to TOP10 Competent Cells (Invitrogen).…”

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats

“…Systemic activity of a foreign protein can be generated by unexpected events such as ectopic transgene expression or via a non-exocrine pathway of the protein from the target tissue to the blood (Rülicke and Hübscher, 2000;Dunn et al, 2005). In this context, investigation of ectopic gene expression in different tissues/organs is an important and unavoidable step to the phenotypic characterization of transgenic animals (Huang et al, 2012;Serova et al, 2012).…”

Copy number and integration sites in growth hormone transgenic goats

“…However, thermal asymmetric interlaced PCR (TAIL-PCR), based on nest PCR and randomly primed PCR category (Liu and Chen, 2007), has become an extremely valuable and versatile tool for detecting insertion sites of foreign genes. TAIL-PCR methods have been successfully used to clone unknown sequences adjacent to known vector sequences in mice (Pillai et al, 2008, Serova et al, 2012 and pig (Zhou et al, 2013); however, there are few reports focusing on transgenic goats because the goat genome has not been completely sequenced.…”