Monosomal Hybrid Technique

Figure 1 depicts the basic technique used for monosomal hybrid generation.[1-3] Donor cells, or human cells in which the separation of chromosomes to a haploid state is sought, are either low-density peripheral blood cells or a transformed lymphoblastoid cell line. Rodent cell lines— typically mouse[3-9] or hamster[2,10-12] cell lines—that have been engineered to allow for the selection of human-rodent fusion progeny are commonly used as recipient cells. Both cell types are initially incubated together in a low volume to promote membrane-to-membrane contact between donor and recipient cells. After a short time, an agent is introduced, such as polyethylene glycol

(PEG)[1,2,6,7,10-12] or electricity,[3-5,8,9] which disrupts cell membrane integrity and promotes fusion. Cells are then washed and allowed to recover from this traumatic treatment for 1-2 days in normal culture media before selective conditions are applied.

Only a small percentage of cells will stably combine into donor-recipient progeny after fusion treatment and the remainder of unfused cells must be eliminated to prevent culture overgrowth. Although many different rodent cell lines are available as recipients, most base their methods of selection on similar biochemical principles. Nucleotides can be synthesized either from basic building blocks by the de novo pathway, or by recycling of preformed bases through the salvage pathway. In the salvage pathway, the enzyme hypoxanthine-guanine phosphoribosyl transfer-ase (HPRT) catalyzes the addition of hypoxanthine or guanine to 5-phosphoribosyl-a-pyrophosphate, forming inosine or guanosine monophosphate, respectively. In recipient cell lines mutated for this enzyme, the salvage pathway is blocked and renders them dependent on the de novo one for growth.[2-12] Alternatively, pyrimidine salvage can also be blocked by mutating the thymidine kinase (TK) gene, which blocks the phosphorylation of thymidine, leading to the formation of thymidine mono-

phosphate.[1]

After fusion, cultures are shifted to a medium containing hypoxanthine, aminopterin, and thymidine (HAT). Aminopterin is a competitive inhibitor of dihydrofolate reductase, which blocks the synthesis of tetrahydrofolate and, consequently, the de novo synthesis of purines. With both synthesis pathways blocked, unfused recipient cells are eliminated, whereas fusion daughter cells can use hypoxanthine and aminopterin in the salvage pathway if they have successfully retained the pertinent genetic material from the human donor cells. Because the gene for HPRT is located on the X chromosome and the TK gene is located on chromosome 17, these methods select directly for progeny containing one or more of these chromosomes, and indirectly for all other human chromosomes retained along with them. Hybrids containing chromosome 2 can be directly selected for using recipient hamster cells in which pyrimidine de novo synthesis is blocked and growth without uridine is impaired.[2] Although other strategies for selection exist, such as temperature-specific defects in protein synthesis,[2] selection systems based

Donor human cell

Recipient rodent cell

Donor human cell

Cell Fusion

Selection

Recipient cells dead Rodent human hybrids proliferate Donor cells dead

Fig. 1 Procedure for the creation of monosomal human-rodent hybrids.

Recipient cells dead Rodent human hybrids proliferate Donor cells dead

Fig. 1 Procedure for the creation of monosomal human-rodent hybrids.

on pathways of nucleotide synthesis are, by far, the most common.

Unfused human donor cells must also be removed from hybrid cultures. Chemicals such as oubain, a disruptor of sodium-potassium pumps,[1] or geneticin, an aminoglycoside related to gentamicin, can be used because human cells are sensitive to their actions.[3] However, the most common strategy exploits the adherent growth characteristics of recipient cells. Lymphoblasts and lymphoblastoid cell lines grow in suspension, but fusion progeny will retain the adherent quality of their recipient parents. Unfused donor cells can then simply be removed from

cultures by repeated washes.[ , ]

After the specific selection criterion is applied, fusion progeny then proliferate to form colonies that are isolated and characterized for their specific human chromosomal complement. During the process of cell culture following fusion, hybrids will eliminate human chromosomes not directly required in the selection process. Over time, human chromosome retention becomes more stable and hybrids can be passaged without further losses.[2] This then has the effect of creating a series of hybrids containing the chromosome directly selected for along with a random assortment of other human chromosomes. Panels of highly polymorphic microsatellites or cytogenetic probes can be then employed to determine the number and allele of each chromosome within a particular hybrid clone. Figure 2 shows such a strategy employed for the analysis of a series of hybrids derived from the fusion of a lymphoblastoid cell line from an individual with trisomy 21 and Ade-C Chinese hamster ovary cells.[10-12] The recipient cells are deficient for the glycinamide ribonucleotide formyl transferase, an enzyme located on human chromosome 21 that blocks purine de novo synthesis. Hybrids containing human chromosome 21 can then be directly selected for by culture in purine-free media.[10-12] Multiple microsatellites are often analyzed to identify markers that are heterozygous and can, therefore, be used to distinguish each individual chromosome in a hybrid series. The original trisomic donor for the series of hybrids shown in Fig. 2 possessed three alleles of 166, 170, and 172 at the dinucleotide microsatellite D21S215 and, consequently, this marker was informative for chromosome 21 content in each hybrid. Although the direct selection methods eliminate nonfused recipient cells and any fusion progeny not containing at least one copy of the selected chromosome, hybrids can still retain more than one copy of that chromosome. In this series, fusion resulted in one hybrid clone retaining one copy of donor chromosome 21 (Fig. 2, row D), one hybrid with two copies of chromosome 21 (Fig. 2, row C), and a third hybrid retaining the complete trisomic set from the donor (Fig. 2, row B). Because the utility of fusion hybrids lies

Fig. 2 Genotype analysis of hybrids at the microsatellite D21S215. DNA from a series of human-hamster hybrids (rows B-D) and the hamster recipient cell line (row A) were amplified at this dinucleotide marker using fluorescent-labeled primers to identify the human chromosome 21 content in each hybrid.

Fig. 2 Genotype analysis of hybrids at the microsatellite D21S215. DNA from a series of human-hamster hybrids (rows B-D) and the hamster recipient cell line (row A) were amplified at this dinucleotide marker using fluorescent-labeled primers to identify the human chromosome 21 content in each hybrid.

with the HNPCC cancer spectrum. The colorectal tumor from the proband exhibited microsatellite instability, which suggested an underlying defect in mismatch repair; however, direct genomic sequencing of the mismatch repair genes MSH2, MLH1, and MSH6 failed to identify a pathologic mutation. Altered Southern blot hybridization patterns for the MSH2 gene were observed for multiple restriction enzyme digestions of genomic DNA from both individuals compared with normal controls. This suggested the presence of a large genomic deletion in the 5' portion of that gene, but attempts to define the deletion

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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