Expression of human lysozyme mRNA in the mammary gland of transgenic mice

Abstract

Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overall microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5' promoter elements of either the bovine beta (line B mice) or alpha s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5.

Expression of a bovine κ-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette

“…The ectopic expression observed in the salivary gland of two of the three lines tested here may be explained by the common structural characteristics of the mammary and salivary glands (Whitelaw et al, 1992). Ectopic expression in the salivary gland has been reported in various transgenic lines using mouse WAP, ovine [3-1actoglobulin, or bovine OLsl-CN mammary gland-specific promoters (Pittius et al, 1988;Whitelaw et al, 1992;Maga et al, 1994).…”

“…DNA from progeny of founder mice was extracted from tail or toe clips of pups as described by Maga et al (1994), and the presence of the transgene was assayed by PCR analysis. Primers JK6 (5'-CAA CTA CCA TGG CAC GTC AC-3') and JK7 (5'-CCG AAG GCA GAC AAC TGG-3'), specific for amplification of a 300bp region of the b-K-CN cDNA, were used.…”

“…Northern analysis was performed as described by Maga et al (1994) on total RNA extracted from mammary gland, kidney, liver, sublingual and submandibular salivary glands, spleen, lung, heart, skeletal muscle, skin, ovary and uterus of day-10 lactating transgenic females. Mammary gland of virgin females and females at day 2 before parturition of days 4, 10 and 17 of lactation were also analysed (n = 3 females per treatment).…”

The production of pharmaceutical proteins from the milk of transgenic animals

“…Some gene constructs containing a given cDNA associated with efficient introns and transcription terminators may however be much less efficient in vivo than the corresponding genomic fragment, although they were significantly more potent in cultured cells (Petitclerc etal, 1995). Introducing the foreign cDNA in the middle of milk protein genes, keeping some of their introns and exons in the cDNA situated before and after proved to be efficient in several cases (Brem et al, 1994;Drohan et al, 1994;Hyttinen et al, 1994;Maga et al, 1994;Brem et al, 1995 (Brem et al, 1994; Platenburg etal, 1994), p-casein (Ebert efal, 1994;Maga et al, 1994), sheep [3-lactoglobulin (Barash et al, 1993Brash et al, 1994;Hurwitz et al, 1994;Platenburg et al, 1994) and rabbit WAP (Bischoff et al, 1992;Devinoy et al, 1994 (Kumar etal, 1994;Stinnakre et al, 1994 (Petitclerc et al, 1995 (Furth etal, 1992), and retroviral vectors (Archer et al, 1994 (Lebas, 1970;Duby et al, 1993), may be attractive, due to the relatively low cost of transgenic generation; rabbit also appears to be appropriate as a transition animal to obtain easily sufficient amounts of recombinant proteins for the study of their biochemical and biological properties before using larger animals to produce these proteins on an industrial scale.…”

“…Since this review was published, additional proteins have been obtained in milk. Among these proteins are human lysozyme (Maga et al, 1994), human insulin-like growth factor-1 (IGF-1) (Maga etal, 1994), bovine chymosin (Brem et al, 1995), human plasminogen activator (Riego et al, 1993;Ebert et al, 1994), human growth hormone (Tojo etal, 1993;Ninomiya etal, 1994), human y interferon (Dobrovolsky etal, 1993), human lactoferrin (Platenburg et al, 1994), human erythropoietin (Hyttinen etal, 1994;Castro et al, 1995), human protein C (Drohan et al, 1994;Paleyanda et al, 1994;Wei et al, 1995), and human albumin (Barash et al, 1993;Hurwitz et al, 1994 (Krimpenfort et al, 1991;Hyttinen etal, 1994 (King and Wall, 1988;Ninomiya et al, 1989). Another method based on the discrimination of DNA methylation in the original insert and in the integrated gene may be used (Cousens et al, 1994).…”