Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.

Nelson, F. Kenneth; Frankel, Wayne N.; Rajan, T. V.

doi:10.1128/mcb.9.3.1284

Cited by 23 publications

(10 citation statements)

References 23 publications

(7 reference statements)

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“…This rate of production of homozygous mutants is similar to that observed for other cultured somatic cells (2,10,12,13). However, since approximately 10% of the homozygous cells will survive these levels of G418 (Fig.…”

Section: Discussionsupporting

confidence: 66%

“…Human cell lines have also been shown to produce homozygous loci from originally heterozygous sites at a low frequency, although the mutational mechanism is not defined (5). In murine lymphoid cell lines, Rajan and coworkers found homozygous cells produced mainly by mitotic recombination; the rate of mitotic recombination was estimated to be 10-5 to 10-6 per generation by fluctuation analysis (10,12,13 …”

Section: Discussionmentioning

confidence: 99%

“…Here, we describe an improved method for obtaining such null mutant cells. Homozygous cells have been known to be spontaneously produced from heterozygous cultured cells by a variety of mechanisms (2,10,12,13). We demonstrate that homozygous cells are routinely produced after initially targeting a single allele and have developed a scheme for the selection of these double knockouts.…”

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confidence: 99%

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Production of homozygous mutant ES cells with a single targeting construct.

Mortensen¹,

Conner²,

Chao³

et al. 1992

Mol. Cell. Biol.

400

261

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We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, %. and c. , T-cell receptor ca, and 1"-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.With the discovery that gene sequences in mammalian cells could be targeted by homologous recombination, new methods for constructing mutationally altered cells that lack particular genes have become available. Over the past several years, a number of groups have demonstrated that heterozygous cells in which one of two alleles has been inactivated can be constructed (4,7,8,14,16,18,19).Production of null mutants by using homologous recombination (in which two copies of the gene have been inactivated) has recently been accomplished in embryonic stem (ES) cells (9, 15) and other types of cells (3). Such mutants should allow the investigation of questions more easily addressed in cell lines rather than in whole animals or tissues. The methods used involved sequential targeting by two separate constructs using two different selectable markers. Here, we describe an improved method for obtaining such null mutant cells. Homozygous cells have been known to be spontaneously produced from heterozygous cultured cells by a variety of mechanisms (2, 10, 12, 13). We demonstrate that homozygous cells are routinely produced after initially targeting a single allele and have developed a scheme for the selection of these double knockouts. MATERIALS AND METHODSConstruction of targeting vectors. In each case, the phosphoglycerate kinase (PGK)-thymidine kinase gene was inserted outside the regions of homology. The production of the a12 targeting construct has been described previously (9). The a43 gene clone was isolated as described for the ti2 gene.A HindIII-SalI fragment was subcloned into Bluescript SK (+) (Stratagene). The construct contained the PGK-neo (neomycin resistance) gene interrupting exon 1 at an NcoI site at the translational start ATG. A 5.6-kb fragment of the murine T-cell receptor a (TCR-a) gene containing all four constant-region exons was subcloned from genomic clone * Corresponding author. X*1.2 (6) into Bluescript SK II (+). The fragment extends from an artificial EcoRI site, 5' of the XbaI site, to the XhoI...

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Section: Discussionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Production of homozygous mutant ES cells with a single targeting construct.

Mortensen¹,

Conner²,

Chao³

et al. 1992

Mol. Cell. Biol.

400

261

View full text Add to dashboard Cite

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“…This gap in our knowledge results from the fact that measuring recombination rates experimentally can be a difficult task, even in model organisms. Nevertheless, many elegant experiments have been done to estimate the number of recombination events per generation in the rDNA of Saccharomyces cerevisiae [e.g., 1 3 10 À2 /generation (Szostak and Wu 1980), 1.3 3 10 À3 (Merker and Klein 2002), and 7.4-7.5 3 10 À5 (Kobayashi et al 2004)] and in the rDNA of murine cells [1.2-1.8 3 10 À5 (Nelson et al 1989)]. Statistical approaches have also been developed to estimate recombination rates indirectly from population genetic data (reviewed in Stumpf and McVean 2003), but these methods need empirical confirmation.…”

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confidence: 99%

Rates of Recombination in the Ribosomal DNA of Apomictically Propagated Daphnia obtusa Lines

et al. 2007

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Ribosomal (r)DNA undergoes concerted evolution, the mechanisms of which are unequal crossing over and gene conversion. Despite the fundamental importance of these mechanisms to the evolution of rDNA, their rates have been estimated only in a few model species. We estimated recombination rate in rDNA by quantifying the relative frequency of intraindividual length variants in an expansion segment of the 18S rRNA gene of the cladoceran crustacean, Daphnia obtusa, in four apomictically propagated lines. We also used quantitative PCR to estimate rDNA copy number. The apomictic lines were sampled every 5 generations for 90 generations, and we considered each significant change in the frequency distribution of length variants between time intervals to be the result of a recombination event. Using this method, we calculated the recombination rate for this region to be 0.02-0.06 events/generation on the basis of three different estimates of rDNA copy number. In addition, we observed substantial changes in rDNA copy number within and between lines. Estimates of haploid copy number varied from 53 to 233, with a mean of 150. We also measured the relative frequency of length variants in 30 lines at generations 5, 50, and 90. Although length variant frequencies changed significantly within and between lines, the overall average frequency of each length variant did not change significantly between the three generations sampled, suggesting that there is little or no bias in the direction of change due to recombination.

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“…4C). The cell culture findings argue strongly against the possibility that the apoB100 in the lipoproteins of the compound heterozygotes might be the result of somatic mosaicism (22,23) or result from somatic recombination between the apoB37 and apoB86 alleles (24,25).…”

Section: Resultsmentioning

confidence: 95%

Reading-frame restoration with an apolipoprotein B gene frameshift mutation.

Linton

Pierotti

Young

1992

Proc. Natl. Acad. Sci. U.S.A.

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We emined a mutant human apolipoprotein B (apoB) allele that causes hypobetalipoproteinemia and has a single cytosine deletion in exon 26. This fameshift mutation was associated with the synthesis of a truncated apoB protein of the predicted size; however, studies in human subjects and minigene expression studies in cultured cells indicated that the mutant allele also yielded a full-length apoB protein. The 1-base-pair deletion in the mutant apoB allele created a stretch of eight consecutive adenines. To understand the mechanism whereby the mutant apoB allele yielded a full-length apoB protein, the cDNA from cells transfected with the mutant apoB minigene expression vector was examined. Splicing of the mRNA was normal; however, 11% of the cDNA clones had an additional adenine within the stretch of eight adenines, yielding nine consecutive adenines. The insertion of the extra adenine, presumably during apoB gene transcription, is predicted to restore the correct apoB reading frame, thereby permitting the synthesis of a full-length apoB protein. In 1979, Steinberg et al. (1) characterized an unusual kindred (the H.J.B. kindred) with asymptomatic familial hypobetalipoproteinemia. Their studies suggested that H.J.B. and two of his siblings, who had extremely low levels of low density lipoprotein (LDL) cholesterol (<8 mg/dl), might be homozygotes for hypobetalipoproteinemia, whereas several other members of the kindred with moderately reduced LDLcholesterol levels (29-62 mg/dl) were postulated to be heterozygotes. In 1987, Young et al. (2, 3) reexamined the H.J.B. kindred and provided evidence for two different defective apolipoprotein B (apoB) alleles: one yielding a truncated apoB, apoB37, and a second yielding very low amounts of a full-length apoB100. They demonstrated that the three family members with extremely low LDL cholesterol levels were compound heterozygotes, whereas subjects with moderately low cholesterol levels were heterozygous for either the apoB37 allele or the allele yielding low amounts of apoB100. Subsequent studies revealed that the mutant allele yielding apoB37 had a 4-base-pair (bp) deletion that resulted in a premature stop codon and a truncated apoB protein containing 1728 amino acids (4).The defect in the second mutant allele yielding very low levels of apoB100 is the subject of this report. The clue that led to the understanding of this allele was the recognition that H.J.B., as well as the other two compound heterozygotes, actually had four bona fide apoB species within their plasma lipoproteins: apoB37, apoB48, apoB100, and apoB86. In this study, we demonstrate that apoB86 and apoB100 are the products of a single mutant apoB allele, which we have designated the apoB86 allele. We show that the apoB86 allele has a 1-bp deletion in exon 26 of the apoB gene and that this frameshift mutation clearly results in the synthesis of apoB86. Furthermore, in cell culture expression studies, the apoB86 allele, which contains a premature stop codon, nevertheless results in the synthesis of a...

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Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.

Cited by 23 publications

References 23 publications

Production of homozygous mutant ES cells with a single targeting construct.

Production of homozygous mutant ES cells with a single targeting construct.

Rates of Recombination in the Ribosomal DNA of Apomictically Propagated Daphnia obtusa Lines

Reading-frame restoration with an apolipoprotein B gene frameshift mutation.

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