Yak fibroblast cell line (Yy) derived from the Datong yak breed (Bos grunniens) was generously provided by Dr. Jikun Wang, Southwest Minzu University, Chengdu, China. The bovine mammary alveolar cell-T (MAC-T, Cc) cell line obtained from Holstein dairy cattle (Bos taurus) was kindly gifted by Dr. Fengqi Zhao, University of Vermont, Burlington, USA. Cells were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, 11965092), supplemented with 10% fetal bovine serum (FBS, Gibco, A5670701), and maintained in 5% CO₂/95% air at 37°C.

To achieve green fluorescence, Lipofectamine 3000 reagent (Invitrogen, L3000150) was used to transfect Yy cells at approximately 70% confluency with eGFP-neo plasmid (Beyotime, D2723-1 μg) in a 24-well plate. The transfection procedures were conducted in accordance with the manufacturer’s protocol. Cells were transfected with 0.5 μg of plasmid per well, and the incubation period was set at 15 min. After 48 h of incubation, Yy cells were examined for green fluorescence using confocal microscopy (Nikon, A1 HD25, Japan).

To achieve mitochondrial staining, a 50 μg MitoTracker stock solution was diluted with 1 mL anhydrous dimethylsulfoxide (DMSO), followed by the addition of DMEM to achieve the desired final working concentration (Table 1). Subsequently, a total of 1 × 10⁷ Cc cells were stained with 300 nM MitoTracker Green FM dye, while both 1 × 10⁷ Cc and Yy cells were stained with 500 nM MitoTracker Deep Red FM dye. All stained cells were incubated for 30 min at 37°C. After incubation, cells were washed twice with prewarmed (37°C) Dulbecco’s phosphate-buffered saline (DPBS) before the addition of prewarmed culture medium.

The culture medium was supplemented with an additional 50 µg/mL of uridine (Solarbio, 58-96-8) and 1 mM of pyruvate (Solarbio, 113-24-6). Subsequently, the mixture was combined with an equal volume of percoll isolation solution (Solarbio, 65455-52-9) and incubated overnight in a cell incubator. After incubation, cell enucleation working solution was prepared by adding 20 μg/mL of cytochalasin B (Solarbio, 14930-96-2). Cc and Yy cells at 80% confluency in a culture dish were collected and resuspended in 25 mL of the working solution in an ultracentrifugation tube (Beckman, 344057). The tube was then placed into an ultracentrifuge (Beckman, XE-100, USA) and centrifugated at 44,000 g, 37°C for 70 min. After centrifugation, a distinct band indicating successful separation could be observed in the solution. This band was carefully transferred to a new 50 mL centrifuge tube, followed by the addition of ten times the volume of culture medium. Then further centrifuged at 5,000 g for 3.5 min. The cells were resuspended in 1 mL pre-warmed DMEM medium and counted by a cell counter (RWD, C100-SE, China). Subsequently, 10⁶ Yy cells were mixed with 10⁶ enucleated Cc cells while 10⁶ Cc cells were mixed with 10⁶ enucleated Yy cells in a 15 mL centrifuge tube. The mixture was then centrifuged at 500 g for 5 min, followed by addition of 100 μL of 50% polyethylene glycol (PEG). After gentle suspension, the mixture was incubated at 37°C for 1 min to facilitate fusion which was subsequently terminated by the addition of culture medium (10 mL). The cells were then cultured in an incubator [26].

Before performing fluorescence-activated cell sorting (FACS), the following types of samples were prepared:

1.

Unstained sample: Cc cells that were not subjected to any staining procedure.

All samples were collected. Following this, 1 mL of pre-warmed DMEM was evenly distributed by blowing and passed through a 40 μm filter into a 5 mL flow tube (BD Falcon, 352235). FACS was performed using FACSAria Fusion instrument (BD, FACSARIA III, USA), which is equipped with four lasers (488 nm, 640 nm, 405 nm, and 355 nm) and 15 detectors. With the inclusion of the BD automated cell deposition unit (ACDU, BD, FACSARIA III, USA) device, cells can be quantitatively sorted into individual wells in a 96-well plate.

Unstained samples were utilized to optimize side scatter (SSC) and forward scatter (FSC) voltages, while the single staining control was employed to exhibit positive signals. MitoTracker Green FM positive cells were detected using a blue laser and fluorescein isothiocyanate (FITC, Invitrogen, M46752) detector, whereas MitoTracker Deep Red FM positive cells were detected using a red laser and allophycocyanin (APC, Invitrogen, M46750) detector (Table 2). To acquire dual-positive cells, the selection of MitoTracker Deep Red-positive cells was based on the presence of MitoTracker Green-positive signal. The dual-positive cells can be sequentially sorted into preheated culture medium-filled wells in 96-well plates, with each well containing only one cell. Dual-positive cells resulting from fusion between Yy cells and enucleated Cc cells were referred to as Yyc cells, while those resulting from fusion between Cc cells and enucleated Yy cells were referred to as Ccy cells.

The cells were cultured in 96-well plates for one or two days. Cell fluorescence was observed and captured using super-resolution confocal laser scanning microscopy (CLSM, A1HD25, Nikon, Japan). This microscope is equipped with six lasers (405 nm, 445 nm, 488 nm, 514 nm, 561 nm, and 647 nm) and multiple fluorescence channels. The appropriate laser wavelength for fluorescence imaging was selected. The subsequent transition to confocal mode facilitated the capture of high-resolution images featuring distinct fluorescence signals. Finally, a scale bar was added to the image before outputting.

After a single cell in the well underwent proliferation and formed a clone, polymerase chain reaction (PCR) identification was performed using 5,000 to 10,000 cells from the clone. PCR was performed using the DirectAmp Animal Tissue PCR Kit (Aidlab, PC5301), where the lysis solution was prepared by combining buffer AD1 and double-distilled H₂O (ddH₂O) in a ratio of 1:9. Each clone received 75 μL of the lysis solution and was thoroughly mixed. The cells were lysed at 98°C for 5 min, rapidly cooled on ice, and then supplemented with 75 μL of buffer AD2. This resulting mixture served as a direct template for PCR amplification. The partial mitochondrial sequence was amplified in a total volume of 25 μL buffer containing 12.5 μL of 2 × DirectAmp PCR Mix, 0.5 μL each forward and reverse primers [27] [10 μM; forward primer: from nucleotide (nt) 682 to nt 701, 5’-GGTCATACGATTAACCCAAG-3’; reverse primer: from nt 1,802 to nt 1,821, 5’-TGGACAACCAGCTATCACCA-3’], along with 4 μL template and 7.5 μL ddH₂O with the following program: 95°C for 5 min, followed by 40 cycles of 94°C for 30 s, 53°C for 30 s and 72°C for 70 s. PCR products were sequenced by Sangon Biotech while validation of mtDNA sequences were performed using SnapGene Viewer software (version 4.1.8).

The technical methodology utilized in this study is depicted in Figure 1, illustrating the approximate approach to obtain the mitochondrial heteroplasmy models.

Based on the intensity exceeding 10³, mitochondrial staining in a single positive cell can be considered indicative of positive staining. As Yy and Cc cells serve as recipients during cell fusion, it is essential to gate the dual-positive signal cells located in the Q2 region of Figure 2. Compared to Model 2, Model 1 generates a higher proportion of fluorescence signals. Subsequently, these gated cells were individually sorted into the 96-well plate in a sequential order, with each well containing only one cell.

The Yyc cell exhibited overall green fluorescence and cytoplasmic red fluorescence, while the Ccy cell displayed cytoplasmic red and green fluorescence that appeared yellow upon superposition (Figure 3). Consequently, both Yyc and Ccy cell represents mitochondrial heteroplasmy with a combination of cattle and yak mitochondria.

After cell lysis, the mtDNA sequence was amplified using PCR. The mtDNA sequences of Cc cells and Yy cells were consistent with previous research [27]. Subsequent sequencing analysis revealed an overlapping peak map, indicating the presence of two distinct types of mtDNA within the Yyc and Ccy cells (Figure 4), thereby confirming mitochondrial heteroplasmy.