Hoon-Taek Lee

Development of a positive method for male stem cell-mediated gene transfer in mouse and pig

Classical approaches for producing transgenic livestock require labor‐intensive, time‐consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994: Proc Natl Acad Sci 91:11298‐11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303‐11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short‐term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate‐buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0–14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7–13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome‐injected testes were all negative for X‐gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3–25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock. Mol. Reprod. Dev. 46:515–526, 1997.

Comparison of maturation, fertilization, development, and gene expression of mouse oocytes grown in vitro and in vivo.

AbstractPurpose: To investigate the difference of in vitro and in vivo grown oocytes, we compared maturation, fertilization, development, and maternal gene expression from both in vitro and in vivo grown mouse oocytes. Methods: The preantral follicles isolated from 12-day-old mice were cultured on Transwell-COL membrane inserts. After culture, maturation, fertilization, and developmental rates were assessed. RT-PCR (reverse transcription—polymerase chain reaction) was performed to examine the expression of β-actin, GDF-9, and IGF-II in matured oocytes. Results: No difference in the nuclear maturation was detected between in vitro and in vivo grown oocytes, but the mean oocyte diameter of the in vitro group was smaller than that of the in vivo group. The fertilization rate was significantly lower in the in vitro group than in the in vivo group (p < 0.05). The capacities of in vitro grown oocyte to cleave and develop to blastocysts were significantly lower than those of the in vivo grown oocytes (p < 0.001). Moreover, blastocyst of in vitro group had fewer total cells than those of in vivo group (p < 0.05). In regards to the expression of genes in mature oocytes, growth differentiation factor-9 (GDF-9) expression was similar between the two groups, but β-actin was significantly reduced in the in vitro group compared to the in vivo group. Particularly, the expression of insulin-like growth factor II (IGF-II) was not found in the in vitro grown oocytes. Conclusions: These results showed that in vitro grown oocytes did not have the same developmental capacity as in vivo grown oocytes. We assume that the aberrant expression of maternal-derived genes in the in vitro grown oocytes may cause the poor embryo viability.

Identification and Classification of Endogenous Retroviruses in Cattle

ABSTRACT The aim of this study was to identify the endogenous retrovirus (ERV) sequences in a bovine genome. We subjected bovine genomic DNA to PCR with degenerate or ovine ERV (OERV) family-specific primers that aimed to amplify the retroviral pro/pol region. Sequence analysis of 113 clones obtained by PCR revealed that 69 were of retroviral origin. On the basis of the OERV classification system, these clones from degenerate PCR could be divided into the β3, γ4, and γ9 families. PCR with OERV family-specific primers revealed an additional ERV that was classified into the bovine endogenous retrovirus (BERV) γ7 family. In conclusion, here we report the results of a genome scale study of the BERV. Our study shows that the ERV family expansion in cattle may be somewhat limited, while more diverse family members of ERVs have been reported from other artiodactyls, such as pigs and sheep.

Effects of Lactation Stage and Individual Performance on Milk cis-9, trans-11 Conjugated Linoleic Acids Content in Dairy Cows.

The goal of this study was to evaluate the effects of lactation stage and individual performance on milk cis-9, trans-11 conjugated linoleic acid (CLA) content in dairy cows. In experiment 1, the milk cis-9, trans-11 CLA content from dairy cows in early (0.33±0.014%), middle (0.37±0.010%), and late stages (0.44±0.020%) showed significant differences (p<0.05); and the individual contents of the major fatty acids, especially cis-9, trans-11 CLA in cows of the same lactation were also variable. In the second experiment design as a validation test, our results once again showed that the individual contents of cis-9, trans-11 CLA were various, and a difference of about 2-fold (0.55% vs 0.95%) was observed, although the animals were offered same diet. These data demonstrated that lactation stage and individual performance have considerable effects on milk cis-9, trans-11 CLA contents.

Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

Cloning and characterization of 5′-untranslated region of porcine beta casein gene (CSN2)

beta-Casein (CSN2) is a major milk protein in most mammals. The CSN2 gene is generally induced by lactogenic hormones bound to its promoter. The expression of this gene can be enhanced by signal transducers and activators of transcription (STAT) and glucocorticoid receptor (GR). Here, we analyzed the promoter and intron 1 regions of the porcine CSN2 gene. The porcine CSN2 promoter and intron 1 regions (-3098bp to +2446bp) were cloned into the pGL3-Basic vector containing the luciferase reporter gene (pCSN2-PEI). Lactogenic signals induced the transcription of porcine CSN2. By using AG490, a Janus kinase (JAK) inhibitor, we demonstrated that STAT5 positively regulates the transcription of porcine CSN2. Further, seven STAT mutants were generated by site-directed mutagenesis. By performing electrophoretic mobility shift assays (EMSAs), we located a critical element for pCSN2-PEI transcription bound to STAT5 in the -102bp to -84bp region. The construct containing only the promoter region (pCSN2-P), however, did not exert any promotive effects on transcription in two cell types-a mouse mammary epithelial cell line (HC11) and porcine mammary gland epithelial cells (PMECs). Thus, the construct containing intron 1 of porcine CSN2 exerts an elevating effect on transcription. We suggest that the transcription of porcine CSN2 is regulated by lactogenic signals via the STAT5 site (-102bp to -84bp) and intron 1.

Effects of Supplementing Brown Seaweed By-products in the Diet of Holstein Cows during Transition on Ruminal Fermentation, Growth Performance and Endocrine Responses

This study was conducted to examine the effects of supplementing brown seaweed by-products (BSB) in the diet of ruminants on ruminal fermentation characteristics, growth performance, endocrine response, and milk production in Holstein cows. In Experiment 1, the effects of different levels (0%, 2%, and 4% of basal diet as Control, 2% BSB, 4% BSB, respectively) of BSB were evaluated at 3, 6, 9, 12, and 24 h in vitro batch culture rumen fermentation. The pH tended to be higher for the higher level of BSB supplementation, with the pH at 12 h being significantly higher (p<0.05) than that of the control. The concentration of ammonia nitrogen was lower at 3, 9, 12, and 24 h incubation (p<0.05) compared with the control, and tended to be low at other incubation times. Volatile fatty acid concentration appeared to be minimally changed while lower values were observed with 4% BSB treatment at 24 h (p<0.05). In Experiment 2, effects of levels (0%, 2%, and 4%) of BSB on growth performance, endocrine responses and milk production were studied with Holstein dairy cows during transition. Dry matter intake, daily gain and feed efficiency were not affected by BSB supplementation. The concentration of plasma estrogen for the control, 2% BSB and 4% BSB after three months of pregnancy were 55.7, 94.1, and 72.3 pg/mL, respectively (p = 0.08). Although the differences of progesterone levels between BSB treatments and the control were minimal, the concentration in 4% BSB treatment increased to 157.7% compared with the initial level of the study. Triiodothyronine and thyroxine levels were also higher after both three months and eight months of pregnancy than the initial level at the beginning of the study. In addition, BSB treatments during one month after delivery did not affect daily milk yield and composition. In conclusion, the present results indicate that supplementation of BSB did not compromise ruminal fermentation, and animal performance at lower levels and hence may have potential to be used as a safe feed ingredient in dairy cows.

Short communication: Retinoic acid plus prolactin to synergistically increase specific casein gene expression in MAC-T cells

Mammary alveolar (MAC-T) cells, an established bovine mammary epithelial cell line, are frequently used to investigate differentiation. A lactogenic phenotype in these cells is induced by treatment with a combination of hydrocortisone, insulin, and prolactin (PRL). The effect of the vitamin A derivative retinoic acid (RA), which induces differentiation in many cells, has not been studied in MAC-T cells. The objective of this study was to evaluate the differentiation potential of RA (1 μM) in MAC-T cells and to examine the effect of combined treatment with RA (1 μM) and PRL (5 μg/mL). Although RA treatment alone inhibited MAC-T cell proliferation, co-treatment of RA with PRL increased cell growth compared with the control group (treated with 1 μg/mL hydrocortisone and 5 μg/mL insulin). The ratio of Bcl to Bax mRNA was decreased in the RA treatment compared with RA+PRL or control. Retinoic acid-induced differentiation of MAC-T cells was associated with an increase in the mRNA expression of αS1-casein (3.9-fold), αS2-casein (4.5-fold), and β-casein (4.4-fold) compared with the control group. Expression of αS1-casein, αS2-casein, and β-casein was increased 12.9-fold, 11.9-fold, and 19.3-fold, respectively, following treatment with RA and PRL combined compared with the control group. These results demonstrate that RA induces differentiation of MAC-T cells and acts synergistically with PRL to increase specific casein gene expression.

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Abstract Effects of extracellular potassium (K+) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whittens medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K+ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K+-free medium. Millimole concentrations of K+ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K+ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K+ was higher than in media with 12 and 16 mM K+. These results suggest that extracellular K+ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K+ in maturation media impair cytoplasmic maturation.

Well-defined differentiation of hesc-derived hemangioblasts by embryoid body formation without enzymatic treatment

Human hemangioblasts exist only during the early embryonic developmental stage thereby limiting the adult cellular source from which to obtain such cells for study. To overcome this, hemangioblast studies have focused on utilizing human embryonic stem cell (hESC) derivatives but current methods are cell-line dependent. Single cell dissociation of a hESC colony quickly led to cell death in most hESC lines due to enzyme treatment which, in turn, reduced induction potential and hemangioblast differentiation efficiency. Therefore, we sought to effectively improve the process of cell dissociation that is adaptable to various hESC lines and increase the initial induction potential of embryoid body (hEB). As a result, we determined an effective cell dissociation method through a comparison study involving various reagents which demonstrated successful dissociation regardless of cell line and enhanced hemangioblast differentiation efficiency.