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. 2015 Mar 4;16(3):4904-17.
doi: 10.3390/ijms16034904.

Secretion of Human Protein C in Mouse Milk

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Free PMC article

Secretion of Human Protein C in Mouse Milk

Chae-Won Park et al. Int J Mol Sci. .
Free PMC article

Abstract

To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.

Figures

Figure 1
Figure 1
Detection of human protein C (hPC) transgene in founder mice by PCR analysis. Four transgenic mice were identified by transgene specific primers. PCR products are placed on the right. Sizes of DNA markers are on the left (in nucleotides). Lane 1: Negative control with DNA; Lane 2–11: Samples extracted from tails. Number 10, 11, 15, and 38 was detected by PCR as expected size. M: Marker.
Figure 2
Figure 2
Transmission into the germline from transgenic mice. DNA was isolated from tail biopsies. DNA samples were digested with KpnI, run on 0.7% agarose gel, transferred to nitrocellulose membranes, and hybridized with hPC cDNA. (A) The second to fifth generations of transgenic mice were amplified by PCR from #10 and #11 lines. Lanes 1: Negative control without DNA; Lanes 2: Negative control with DNA; Lane 3: Positive control with hPC cDNA vector; Lanes 4–7: #10 line; Lanes 8–11: #11 line; (B) Southern blot results from heterozygotes DNA samples. Number 4–7: #10 lines; Number 8–11: #11 lines; (C) Southern blot results in the fifth generation of homozygous Tg mice were analyzed from #10 and #11 lines. Electrophoresis results from KpnI enzyme cut (left). Number 2–3: #10 line; Number 4–5: #11 line. V: Constructed vector; Ne.: Negative Control. M: Marker.
Figure 3
Figure 3
mRNA expression by RT-PCR, qRT-PCR, and Northern blot. (A) RT-PCR results from mammary glands and other tissues; (B) qRT-PCR results from mammary glands on days 1, 7, and 15 during lactation; (C) Northern blot probed with hPC cDNA in the #10 line. M: marker; He: heart; Li: liver; Ki: kidney; Sp: spleen; Ne: negative control.
Figure 4
Figure 4
Western blot analysis of hPC from the mammary glands and milk during lactation. (A) Protein detection from mammary glands during lactation; (B) The quantity analysis of hPC expressed in the mammary glands; (C) hPC protein detection assessed in the milk of the #10 and #11 lines via Western blot. SC: single-chain hPC forms; HC: heavy-chain hPC form; LC: light-chain hPC forms; CHO: hPC expressed into the CHO cells transfected with hPC cDNA.
Figure 5
Figure 5
Localization of hPC protein expressed in the mammary glands during lactation by immunohistochemistry and in the cultured Tg mice mammary gland cells by immunofluorescence. (A) Immunohistochemistry. Representative immunohistochemical analyses using hPC antiserum (1:1000) and swine anti-rabbit secondary antibodies (1:1000). Preimmune serum (1:1000) was used for primary antiserum as the negative control. Mammary gland sections are shown on days 1, 7, and 15 during lactation. Black bar = 100 μm. Arrows indicate alveolar epithelial cells of the mammary gland; (B) Immunofluorescence in cultured mammary glands of Tg mice (#10 line) on day 7 during lactation. Counter staining was performed with DAPI (blue). hPC expression was detected by Alexa 488 (green). The merged picture shows blue and green colors.
Figure 6
Figure 6
The quantity of rec-hPC in the Tg mice milk by ELISA. (A) rec-hPC in the #10 lines analyzed by ELISA on days 1, 7, and 15 during lactation; (B) hPC analysis in the #11 line. Each point and vertical bar represents the mean and standard error of the mean (SE) values, respectively, for the three different samples.

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