• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.17-17
• /
• 2003

The milk of transgenic animals may provide an attractive vehicle for large-scale production of hEPO. Since glycosylation is cell type specific, recombinant human EPO (rhEPO) produced in different host cells contain different patterns of oligosaccharides, which could affect the biological functions. However, there have been no reports on the characteristics of rhEPO derived from milk of transgenic animals. To address this objective, several transgenic mice by using pWAPhEPO and/or pBC1hEPO expression vector were produced. However, 2 lines of pWAPhEPO founder female mouse died during late gestational day (day 18) before offspring could be obtained. They showed a severe splenomegaly, Unlike those of pWAPhEPO, mammary gland epithelial cells from biopsies of lactating pBC1hEPO transgenic mice had marked immunoreactivity to EPO and any activity was not detected in other tissues. The expression level of rhEPO is about 0.7% of mammary gland cellular total soluble proteins and an amount of 300~500 mg/L rhEPO is secreted into milk. Furthermore, the pBC1hEPO transgenic mice transmitted this character to their progeny in mendelian manner. In order to determine the extent of glycosylation variation, N-linked oligosaccharide structures present in the milk-derived rhEPO were characterized. Most of milk-derived rhEPO is fully glycosylated. the biological activity of milk-derived rhEPO was comparable to that of purified CHO-derived rhEPO, and milk-derived rhEPO showed relatively stable after freezing and thawing. Taken together, the results illustrate the potential of transgenic animals in the large-scale production of biopharmaceuticals.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.4
• /
• pp.462-466
• /
• 2001

To understand molecular mechanisms of mouse mammary gland involution, clones were isolated by differential screening of a cDNA library. Partial sequences of a clone showed 100% identity to cDNA sequences of mouse lysozyme P gene. Northern analysis was performed to examine expression levels of lysozyme mRNA in mammary gland at several physiological states. Expression of lysozyme gene was induced at involution day 5 compared with lactating stage. High levels of lysozyme mRNA were also detected at virgin tissues. Two types of separate genes, P and M lysozyme, have been known in mouse, and we found that both lysozyme P and M genes were expressed in mammary tissues by reverse transcriptase-polymerase chain reaction. The lysozyme enzyme activity determined by lysoplate assay was also higher in involuted mammary tissues compared with lactating tissues, showing a similar trend to its mRNA levels. Lysozyme is an antimicrobial protein and involved in host defense mechanism. The increase in lysozyme gene expression may help to prevent microbial infection during mammary gland involution at which stage the residual milk in the mammary gland provides good nutritional sources for microbial growth.

• Proceedings of the Korea Environmental Mutagen Society Conference
• /
• 2003.10a
• /
• pp.109-110
• /
• 2003

• Parasites, Hosts and Diseases
• /
• v.27 no.2
• /
• pp.79-86
• /
• 1989

A pathogenic free-living amoeba, Naegleria fowleri, causes primary amoebic meningoencephalitis to human and experimental animals. This infection is rare, but the mortality is very high. Nowadays, drug treatment or active immunization of human or mice are being tried with partial effectiveness. This study shows passive immunization effect by transfer of immunized spleen cells, serum, or milk from immunized mother in mouse experimental model. Young BALB/c mice were immunized intraperitoneally with $2~3{\times}10^{6}$ trophozoites of N. fowleri, and spleen cells and sera were collected for injection to recipient mice. There were seven transfer groups, i.e., immunized mouse serum, spleen cells, serum and spleen cells, normal mouse serum, spleen cells, serum and spleen cells, and control group. Three days later, BALB/c mice were inoculated with $1{\times}10^{4}$ trophozoites of N. fowleri intranasally. After infection, decreased mortality ana prolonged survival time of mice were noted in immunized Bloops compared with non.immuniBed control group. The groups Injected with immunized spleen cells or normal serum shewed lower moltality than that of controls bult showed no changes of Serum IgG level. The groups injected with immunized serum or normal spleen cells showed increased serum IgG level after immunization but hundred percent mortality was observed. Mother mice were ifnfnunised increperitqneeliy with $2~3{\times}10^{6}$ trephozoites of N. fowleri at the end of pregnancy and weaning Period. Soon after the delivery, Jitters born of non-immunszed mother were matched with immunized mother for feeding immune milk. After three weeks, the litters were infected with $1{\times}10^{4}$ trophozeites of N. fowleri or sacrificed for serum collection to measure the IgG levels. The results show that anti-JV. fowleri IgG from mother was transferred to litter through milk but this IgG did not inauence the mortality or survival time of the infected mice.

• Food Science of Animal Resources
• /
• v.25 no.2
• /
• pp.250-256
• /
• 2005

To investigate the effect of bovine colostral whey fractions on in vitro proliferation of mouse splenocytes, polypeptide fractions were separated from acid whey into 3 fractions depending on molecular weight by ultrafiltration: Fraction I, which contains the polypeptide larger than 10,000 Da, Fraction n, which contains the polypeptide ranging from 1,000 Da to 10,000 Da and Fraction III, which contains the polypeptide smaller than 1,000 Da. Fraction II showed the highest proliferative effect of mouse splenocytes among the colostral whey fractions and this proliferative activity increased in dose dependent manner. Unheated Fraction II and Fraction III showed significantly (p<0.01) higher proliferative effects than others but heated Fraction II showed the highest enhancing effect of mouse splenocyte among heated whey fractions (p<0.01). The supplementation of Fraction II and Fraction m showed greater proliferative effect of mouse splenocytes stimulated by concanavalin A (Con A) than that of whole whey or Fraction L Proliferative effect of mouse splenocytes stimulated by phytohemagglutinin (PHA) was the highest when Fraction II was supplemented Proliferative effect of the colostral whey fractions on mouse splenocytes by stimulation of lipopolysaccharide (LPS) was markedly enhanced by supplementation of Fraction II and Fraction m compared with whole whey and Fraction L It was estimated that colostral whey fraction containing IGF-I positively affected proliferation of mouse splenocyte.

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.6
• /
• pp.743-749
• /
• 2004

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

• Reproductive and Developmental Biology
• /
• v.32 no.1
• /
• pp.45-49
• /
• 2008

We have previously produced transgenic (TG) mice expressing the human lactoferrin (hLF), interleukin-10 (hIL-10), and thrombopoietin (hTPO) proteins in the milk. In this study, we examined whether simple crossbreeding between two kids of a single transgenic mouse can produce double transgenics co-expressing two human proteins.. The hLF male, and the hIL-10 male were crossbred with the hIL-10 and hTPO females, and the hTPO female, respectively. PCR analysis for genotyping showed 32%, 23% and 24% double transgenic rates for hLF/hIL-10, hLF/hTPO, and hIL-10/hTPO transgenes, respectively. We analyzed the expression levels of the human proteins from double transgenic mice and compared those with their single transgenic siblings. All double transgenic co-expressed two human proteins at comparable levels to singles', unless hTPO was not co-expressed: for hLF, 1.1 mg/ml in hLF/hIL-10, whereas 0.5 mg/ml in hLF/hTPO; for hIL-10, 4.1 mg/ml in hIL-10/hLF, whereas 1.4 mg/ml in hIL-10/hTPO. Ihe downregulation of hTPO to half level of singles' was observed in double transgenic mice. The possible reason why hTPO co-expressed might lead to down-regulation of another human protein was discussed. These results suggested that double transgenic generated by crossbreeding between two singles' could be useful system for bioreactor.

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.212-212
• /
• 2004

Collagen has been widely studied for medical applications. Previous studies have shown that the bovine β-casein promoter were able to drive cell-specific and hormone-dependent expression to a mouse mammary cell line but failed to induce accurate expression to the mammary gland. of transgenic mice. (omitted)

• Korean Journal of Animal Reproduction
• /
• v.17 no.4
• /
• pp.375-383
• /
• 1994

The human growth hormone (hGH) gene uder the control of the rat $\beta$-casein promoter gene was designed to produce transgenic mouse expressed hGH gene in only mammary gland. One hundred seventy two eggs microinjected were transferred to the oviducts of pseudopregnants and 43 offspring were delivered. By Southern blotting hybridization, 3 were transgenic with rat $\beta$-casein/hGH gene. The copy numbers of three transgenic founder were 1, 5, and 15, respectively. A radioimmunoassay was developed to quantitate the amount of expression of the hGH gene in mammary gland of transgenic mice. The amount of hGH was 13.3ng/ml in the lactating milk of one transgenic line, showing predominantly higher than 3.0ng/ml in milk of control mice. Therefore, our findings suggested that $\beta$-casein promoter may induce the tissue specific expression of structural gene.

• Development and Reproduction
• /
• v.13 no.3
• /
• pp.191-198
• /
• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.