The construct to generate the transgenic mice was produced by inserting human LY6K cDNA, which was previously described (22), into the pMAMneo vector (Clontech Laboratories, Inc., Mountainview, CA, USA). The full-length human LY6K cDNA fragment was amplified by polymerase chain reaction (PCR) under the following thermocycling conditions: Initial denaturation at 98°C for 5 min, 35 cycles at 98°C for 10 sec, 50°C for 50 sec and 72°C for 20 sec; and final extension at 72°C for 5 min. For PCR, Taq Polymerase, dNTPs and reaction buffers (Real Biotech Corporation, Taipei, Taiwan) were used according to the manufacturer's protocol. Specifically-designed primers containing NheI or XhoI restriction enzyme sites were used and the sequences were as follows: Forward, 5′-CTAGCTAGCATGGCGCTGCT-3′; and reverse, 5′-GGCCTCGAGTCAAGACAGGC-3′. Fragments digested with NheI and XhoI restriction enzymes (New England Biolabs, Ipswich, MA, USA) were purified by the HiYield Gel/PCR extraction kit (Real Biotech Corporation) according to the manufacturer's protocol, and then inserted into the corresponding restriction sites of the vector. The recombinant construct carrying the transgene was microinjected into the pronuclei of fertilized eggs of B6 mice (Animal Facility, Sookmyung Women's University, Seoul, Korea), and the eggs were then transferred into the oviducts of pseudo-pregnant female mice. Transgenic founder mice were bred with B6 mice to establish transgenic lines; a total of 7 founder mice (8–9 weeks-old; average weight, ~20 g) were generated. All mice were housed in a specific pathogen-free barrier facility with a 12 h light/dark cycle and maintained at a humidity of 40–60% at 23°C. Mice had ad libitum access to food and water. After 6 weeks, mice were euthanized by cervical dislocation for further analysis. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Sookmyung Women's University (Seoul, South Korea).

Genomic DNA was extracted from the tail biopsies of each mouse using a Quick-DNA^™ Universal Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. To confirm the presence of the transgene, PCR was performed to amplify specific fragments of the hLY6K gene. The sequences of the primers used were as follows: Forward, 5′-TGTGGTTTAGGTTGGAGTGTAGTG-3′; and reverse, 5′-CTCATCAAAAAAATCTCCCCAAC-3′. EmeraldAmp^® GT PCR Master Mix (Clontech Laboratories, Inc.) was used to amplify DNAs according to the manufacturer's protocol. The thermocycling conditions were as follows: Initial denaturation step for 2 min at 95°C, 30 cycles for 30 sec at 95°C, 30 sec at 58°C and 30 sec at 72°C, and the final extension step for 5 min at 72°C. The amplified DNA samples were resolved on a 1.0% agarose gel and separated by electrophoresis at 200 V for 40 min, which produced bands of 360 bp. To compare the amount of the transgene, standard samples containing 1, 10 and 100 copies of the transgene were prepared and mixed with the genomic DNA of wild-type B6 mice, then amplified and separated on the same agarose gel.

To examine the copy number of the transgene, a standard curve was generated using 1, 10 and 100 copies of the transgene diluted in the genomic DNA of wild-type mice. qPCR was performed using SYBR-Green qPCR Master Mix (PCR Biosystems, Ltd., London, UK), and each reaction sample contained the following components: 100 ng genomic DNA, 10 µM of each primer and 5 µl SYBR Green qPCR Master Mix. The sequence of the primers used were as follows: hLY6K forward, 5′-AGCCCATGCCCTTCTTTTACCTCA-3′; and hLY6K reverse, 5′-CCAGCCACAGCCCACCACAG-3′; mouse Gapdh (mGapdh) forward, 5′-GCTGAGTATGTCGTGGAGTC-3′; and mGapdh reverse, 5′-ATGGACTGTGGTCATGAGC-3′. The mGapdh gene was amplified as an endogenous control. qPCR cycling conditions were as follows: A pre-incubation step of 10 min at 95°C; followed by 45 amplification cycles of 10 sec at 95°C, 10 sec at 60°C, and 10 sec at 72°C; and a final elongation step of 1 min at 65°C. The standard curve was determined by plotting ΔCq (ΔCq=Cq_hLY6K-Cq_Gapdh) against the log of hLY6K gene copies of the corresponding standard samples, and the copy numbers of the transgene in transgenic mice were determined by the 2^−ΔΔCq method (23). The formula was as follows: 2^−ΔΔCq=2^{−(ΔCq of transgenic samples-ΔCq of wild-type samples)} (24).

RNAs were extracted from each tissue of 3 mice per group (wild-type and transgenic mice) using a NucleoSpin^® DNA/RNA/Protein kit (Machery-Nagel GmbH, Düren, Germany), according to the manufacturer's protocol. RNAs (2 µg) diluted in 12.5 µl of sterile RNase free water were reverse transcribed to cDNAs using an M-MLV Reverse Transcription kit (Promega Corporation, Madison, WI, USA) at 42°C for 1 h and then inactivated at 70°C for 10 min.

Following RT, as described above, cDNA samples (100 ng) were used as templates to amplify specific fragments with RBC Taq Polymerase, and each reaction mixture contained the following: 3 µl 10X RBC Reaction Buffer, 3 µl dNTPs (2.5 mM), 10 mM of each primer, 100 ng of cDNA, 1 µl RBC Taq Polymerase and 21.7 µl double-distilled water. The sequences of the primers used to detect hLY6K or mGapdh were as follows: hLY6K forward, 5′-TGCTCGCCTTGCTGCTGGTC-3′; hLY6K reverse, 5′-TCGCTGCACAACCAGCGGAG-3′; mGapdh forward, 5′-CGGTGCTGAGTATGTCGTGGAG-3′; and mGapdh reverse, 5′-TGTCATCATACTTGGCAGGTTTC-3′. Amplification condition were as follows: Initial denaturation step for 2 min at 95°C, 30 cycles for 30 sec at 95°C, 30 sec at 58°C and 30 sec at 72°C; and the final extension step for 5 min at 72°C. Following amplification, the PCR products were separated on 1.0% agarose gels by electrophoresis at 200 V for 40 min.

To further compare the RNA expression level of hLY6K in wild-type and transgenic mice, RT-qPCR was conducted using SYBR Green qPCR Master Mix (PCR Biosystems, Ltd.). cDNA samples (100 ng) were used as templates, and the sequences of the primers to detect hLY6K and mouse Gapdh were same as those used to examine the transgenic copy number. qPCR cycling conditions were as follows: A pre-incubation step of 10 min at 95°C; 45 amplification cycles of 10 sec at 95°C, 10 sec at 60°C and 10 sec at 72°C; and a final elongation step of 1 min at 65°C. The relative expression levels of hLY6K were calculated by the 2^−ΔΔCq method, normalized against the level of mGapdh.

To obtain proteins from the mouse tissues, tissues was disrupted and homogenized with lysis buffer RP1 (Macherey-Nagel GmbH & Co., Düren, Germany) containing 1% β-mercaptoethanol (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and stainless steel beads using TissueLyser (Qiagen GmbH, Hilden, Germany) at 1500 oscillations/min for 1 min at room temperature. Protein extracts were prepared using a NucleoSpin^® DNA/RNA/Protein kit and buffers included in kit (Macherey-Nagel GmbH & Co.), according to the manufacturer's protocol. Protein extracts were dissolved with Protein Solving Buffer containing the reducing agent TCEP (Macherey-Nagel GmbH & Co.) and boiled at 98°C for 3 min. The protein concentration was measured using a bicinchoninic acid solution and copper (II) sulfate solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Samples were loaded (30 µg/lane) and separated by SDS-PAGE (10–12% gel) and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk in PBS containing 0.2% Tween-20 for 1 h at room temperature. Immunoblotting was conducted using an anti-Ly6K antibody (cat no. sc-87282; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for the detection of human and mouse Ly6K, and an anti-β-actin antibody (cat no. A300-491A; Bethyl Laboratories, Montgomery, TX, USA), which were diluted at 1:1,000 in 1% skim milk in PBS containing the detergent 0.2% Tween-20 overnight at 4°C. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat no. ADI-SAB-300-J; Enzo Life Science, Inc., Farmingdale, NY, USA) was used and diluted at 1:4,000 in 2% skim milk in PBS containing the detergent 0.2% Tween-20 for 1 h at room temperature. Immunoreactive proteins were detected using the enhanced chemiluminescence reagent EzWestLimiplus (cat no. WSE-7120; ATTO Corporation, Tokyo, Japan).

Cell populations were isolated from the thymus or spleen of the transgenic mouse and investigated by flow cytometry. Cells were washed with 1X PBS and then fixed with 70% ethanol overnight at 4°C. To examine the expression of surface markers of thymocytes, cells were stained with specific antibodies targeting surface markers of lymphocytes: Conjugated PerCP-Cy5.5-anti-CD44 (cat no. 45-0441-82; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), conjugated allophycocyanin (APC)-anti-CD25 and conjugated APC-anti-B220 (cat nos. 17-0251-82 and 17-0452-82; all from Invitrogen; Thermo Fisher Scientific, Inc.), conjugated PE-anti-CD4 and conjugated fluorescein isothiocyanate-anti-CD8 (cat nos. ab134354 and ab28010; all from Abcam). All antibodies were diluted 1:200 in FACS buffer (1X PBS containing 1% BSA) and incubated with each sample for 30 min on ice. Analysis was conducted using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using FlowJo 7.6.5 software (Tree Star, Inc., Ashland OR, USA).

All experiments were repeated three times independently. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). The data are presented as the mean ± standard deviation. The RT-qPCR data were analyzed by Student's t-test, and the other data were examined by a two-way analysis of variance followed by a Bonfferoni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of human LY6K on mammary development in vivo, transgenic mice overexpressing the human LY6K gene were established. We generated transgene construct for the full-length human LY6K gene downstream of the MMTV-long terminal repeat (MMTV-LTR) promoter (Fig. 1A). The transgene was injected into fertilized mouse eggs, which were then transferred into the oviducts of female mice. The potential founder mice were crossed with C57BL/6 (B6) mice, and 7 transgenic mouse lines were generated. Genomic DNAs extracted from the transgenic mice were amplified by PCR with human LY6K-specific primers. As shown in Fig. 1B, 4 of the 7 transgenic mice carried the hLY6K transgene. To further examine the copy number of the hLY6K transgene, qPCR was performed, revealing that transgenic mouse B had the highest expression of the transgene (Fig. 1C), and this was used for further study.

To determine the tissue specificity of the transgene expression, the RNA levels of hLY6K were examined in wild-type and transgenic mice by RT-PCR (Fig. 2A). Tissues were harvested from the thymus, spleen, mammary gland and other organs of 6-week-old virgin mice, and RNAs were extracted from each tissue sample, which were subsequently analyzed by RT-PCR. To avoid the detection of mouse LY6K instead of human LY6K, their sequences were compared using NCBI nucleotide BLAST, and there was no significant similarity except for the Ly-6/uPAR domain [(25), https://blast.ncbi.nlm.nih.gov/Blast.cgi; human LY6K gene accession number, NM_017527; mouse LY6K gene accession number, NM_029627]. Thus, PCR primers were designed to recognize only human LY6K mRNA but not the endogenous mouse LY6K, and it was confirmed that hLY6K-specific primers only amplified LY6K mRNAs extracted from human cell lines but not those from mouse cells (data not shown). Amplified products were separated on the agarose gel. As Fig. 2A illustrates, the mRNA expression of hLY6K was detected strongly in the thymus and weakly in the spleen and lung, while other tissues, including the mammary gland, displayed no expression of the transgene. For further examination, RT-qPCR was performed to quantitatively compare the RNA level of hLY6K in the wild-type and transgenic mice. RNAs extracted from the thymus and spleen were examined and, as indicated in Fig. 2B, the relative RNA expression of hLY6K was markedly increased in the thymus and spleen of the transgenic mouse compared with those of the wild-type mice. Furthermore, western blot analysis revealed that the transgenic mouse had markedly higher protein levels of Ly6K in the thymus and spleen compared with those of wild-type mice (Fig. 2C). These results indicated that the hLY6K transgene with the MMTV promoter was expressed specifically in the thymus and spleen, rather than in other tissues, including the mammary gland.

As previously stated, the hLY6K transgene was significantly expressed in the spleen of the transgenic mouse examined. The spleen filters and generates blood cells; however, it is also involved in lymphocyte proliferation and activation as one of the major sites where blood-circulating antigens are presented to lymphocytes in order to generate an immune response (26). Therefore, the present study investigated whether the overexpression of hLY6K caused any changes to lymphocytes in the spleen. First, the distribution of T cells was verified according to the expression of CD4 and CD8, the surface markers of mature T cells. As shown in Fig. 3A, including helper T cells (CD8⁻CD4⁺) and cytotoxic T cells (CD8⁺CD4⁻), the proportion of each cell type did not exhibit any significant change in the spleen compared with the levels in wild-type mice. B cell populations in the spleen were then investigated by analyzing the expression of B220, a murine B cell marker, and CD25, the α chain of the IL-2 receptor (27) (Fig. 3B). Normally, CD25 expression is characteristic of CD4⁺FoxP3⁺ regulatory T cells in mice and only 2% of B cells express CD25 in the spleen (28). As shown in Fig. 3B, there was no significant change in the proportion of B220⁺ and CD25⁻ B cells between the wild-type and transgenic mice. These findings implied that the overexpression of hLY6K did not have a significant effect on lymphocyte development in the spleen.

The expression of hLY6K was detected in the spleen and in the thymus (Fig. 2). The thymus is a specialized lymphoid organ where T cell development occurs. Therefore, the present study investigated whether hLY6K overexpression influenced T cell development in the transgenic mouse. DN naïve T cells, which are negative for CD4 and CD8 expression, can be subdivided into four subgroups (DN1-DN4) depending on their expression of CD44 and CD25 (29). Flow cytometric analysis was conducted to examine the expression of CD44 and CD25 in thymocytes isolated from wild-type and transgenic mice, and cells were categorized depending on the expression of those markers. Fig. 4A demonstrates the distribution of early naïve T cells in the thymus of wild-type and transgenic mice. The present study identified that the proportion of the DN2 (CD25⁺CD44⁺) subgroup was decreased markedly in the transgenic mouse compared with wild-type mice, whereas the DN1 (CD25⁻CD44⁺) and DN3 (CD25⁺CD44⁻) populations exhibited no significant differences. Notably, the DN4 (CD25⁻CD44⁻) population was markedly increased in the transgenic mouse, suggesting that more naïve T cells had completed TCR rearrangement and had begun to express CD4 and CD8 molecules for the next stage of development. Therefore, the levels of CD4 and CD8 (surface markers of mature T cells) were examined in the thymic T cells of each type of mouse to compare further developmental steps (Fig. 4B). The results revealed that the double positive (CD4⁺CD8⁺) subgroup was increased in the transgenic mouse, as hypothesized. However, the proportions of cytotoxic T cells (CD8⁺CD4⁻) and helper T cells (CD8⁻CD4⁺) were decreased in the transgenic mouse. This implied that fewer T cells were fully differentiated in the transgenic mouse, when hLY6K was overexpressed, compared with the wild-type mice, despite an increased number of lymphocytes undergoing TCR rearrangement.