**2.4. DNA content**

Measurement of total or nuclear DNA content is used in characterization of somatic cell hybrids, but is not necessarily intended as an identity test for authenticating such cells. The excess number of chromosomes present in certain cell hybrids relative to the parental cells (discussed above) is also reflected in an increase in DNA content in the hybrid cells. This increase may be detected using microspectrophotometric analysis for nuclear DNA [38] or flow cytometric analysis of propidium iodide-stained cells for total DNA [32]. For instance, while authenticating a series of human × bovine hybrid cell lines, van Olphen and Mittal [32] found that the total DNA content of the hybrid cells was 51–77% greater than the average value for the parental cells (**Table 1**).

Levels of nuclear DNA in bovine × mouse hybrid cells corresponded to the relative increases in chromosome count for the hybrids [38]. A hybrid with mean chromosomal count of 53 (vs. parental values of 44 and 44) was found to have nuclear DNA content similar to the parental mouse cell, while a hybrid with mean chromosome count of 89 displayed a bimodal nuclear DNA content, with one peak similar to that of the parental mouse cell and another peak at around twice the parental cell peak value [38].

hybrid cells in which one of the parental cells was mouse, as observed also by Attardi and Attardi [47], diminishes the utility of the nested PCR method for identifying inter-species cell

**Figure 3.** Use of nested PCR to evaluate a human × mouse hybrid cell line [4G12] and a mouse × rat hybrid cell line [N18-RE-105]. Multiplex group 1 (targeting human, mouse, rat, rabbit, cat, cow, and pig) or the corresponding speciesspecific primer pairs were used. Note that only the mouse DNA is detected in these two hybrid cells (from [48]).

Authenticating Hybrid Cell Lines

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http://dx.doi.org/10.5772/intechopen.80669

Chan et al. [49] used STR profiling to identify presumed intra-species human hybrid cell lines comprised of HeLa × EBV-negative NPC (Epstein-Barr virus-negative nasopharyngeal carcinoma) cells. Four EBV-negative NPC cell lines (CNE-1, CNE-2, HNE-1, and HNE-2) were found to have STR profiles similar to each other, and also shared at least one allele with HeLa across 16 STR loci, as well as additional alleles at several of the STR loci that were attributed to an unknown EBV-negative NPC cell. High-throughput RNA sequencing by Strong et al. [50] resulted in similar conclusions and raised similar concerns for three other EBV-negative NPC

Because these particular EBV-negative NPC cell lines have additional alleles, they do not satisfy the usual match criteria for human cell line authentication [51]. However, it is important to consider all available evidence when deciding if cross-contamination has occurred. The data from Chan et al. [49] showed that CNE-1, CNE-2, HNE-1, and HNE-2 carry an allelic variant (D13S317 13.3) that is characteristic of HeLa derivatives [51]. Strong et al. [50] showed that CNE-1, CNE-2, HONE-1, AdAH, and NPC-KT carry human papillomavirus 18 (HPV18), which is an unexpected finding for NPC cell lines, and display viral and cellular genomic rearrangements that are consistent with HeLa. Looking at all the evidence, it is reasonable to conclude that these EBV-negative NPC cell lines represent not simply cross-contamination

hybrids.

*2.5.2. STR profiling*

cell lines (HONE-1, AdAH, and NPC-KT).

with HeLa, but rather somatic cell hybridization with HeLa.

Both total DNA and chromosome count for a hybrid cell may evolve with continued passage of a culture, due to the propensity for loss of chromosomes derived from one or both parental cells, as mentioned above.

#### **2.5. Nucleic acid sequence-based methods**

The ability to detect intra-species and inter-species cell hybrids has been greatly facilitated by the development of nucleic acid sequencing methods, such as DNA barcoding (PCR- or sequencebased approaches targeting mitochondrial genes), STR analysis, and next generation sequencing.

#### *2.5.1. DNA barcoding*

Ono et al. [48] used a nested PCR targeting the cytochrome b gene of 7 animal species (human, mouse, rat, rabbit, cat, cow, and pig) to authenticate two inter-species hybrid cell lines. These included the 4G12 hybridoma cell line (human B lymphocyte × mouse myeloma) and the N18-RE-105 hybridoma cell line (mouse glioma × rat neural retina). Cytoplasmic isoenzymes from the two hybridoma cell lines were found to display human- or rat-specific migration patterns in isoenzyme analysis, but yielded a result expected for mouse in the nested PCR (**Figure 3**). The authors concluded that the preferential retention of mouse mitochondria in

**Figure 3.** Use of nested PCR to evaluate a human × mouse hybrid cell line [4G12] and a mouse × rat hybrid cell line [N18-RE-105]. Multiplex group 1 (targeting human, mouse, rat, rabbit, cat, cow, and pig) or the corresponding speciesspecific primer pairs were used. Note that only the mouse DNA is detected in these two hybrid cells (from [48]).

hybrid cells in which one of the parental cells was mouse, as observed also by Attardi and Attardi [47], diminishes the utility of the nested PCR method for identifying inter-species cell hybrids.
