Targeted integration of the Ren-1D locus in mouse embryonic stem cells.

Miller, Christopher C.J.; McPheat, Jane; Potts, Wendy

doi:10.1073/pnas.89.11.5020

Cited by 21 publications

(7 citation statements)

References 33 publications

(37 reference statements)

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“…6C) [14,18] (6). One possibility for the discrepancy between our results and those cited above (ii, 23,31) is that the latter case involved assays that looked specifically at classical crossing-overs with two homologous junctions, one on each side of the integrated vector. Classical crossing-overs were not observed in the present study.…”

contrasting

confidence: 53%

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Richard¹,

Belmaaza²,

Gusew³

et al. 1994

Mol. Cell. Biol.

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Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (LI) elements as substrates. We showed previously that an exogenous Li element could readily acquire endogenous Li sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous Li elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous Li element into an endogenous Li element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous Li elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous LI elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous Li elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.Mammalian cells contain numerous nonallelic repeated sequences. These originate from multicopy genes (e.g., rRNA, small nuclear RNA, and histone genes), from gene families (e.g., the immunoglobulin, HLA, and globin genes), and from repeated elements (e.g., LINEs, SINEs, and minisatellites). A number of reports have shown that nonallelic repeated sequences can undergo homologous recombination. These homologous recombination events have been associated with gene function (20), certain genetic disorders (15,16,21,22,25), and genome evolution (3,12).One common feature of repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using the LINE-1 (long interspersed nuclear elements or Li) repeated elements as substrates. The Li family of interspersed repetitive elements is ubiquitous in mammals (17). Their number is in the order of 105 copies dispersed throughout the genome, such that the density of Li elements is of about one copy per 30 to 60 kb of genomic DNA (14, 18). Li elements are estimated to account for 5 to 10% of mammalian genomes. A full-length Li element is 6 to 7 kb long, but over 90% of Li elements are truncated at the 5' end. In a given species, there is a very large spectrum of homology between individual copies that can range from 99% t...

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contrasting

confidence: 53%

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Richard¹,

Belmaaza²,

Gusew³

et al. 1994

Mol. Cell. Biol.

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“…Shen and Huang (29), who coined the term MEPS (minimal essential processing segment) to describe the threshold length of uninterrupted homology required for efficient recombination, argue that the rate of 6664 SCHEERER AND ADAIR recombination between any two sequences should be proportional to the number of MEPS units that it contains, and hence a linear relationship between length of homology and recombination frequency is expected. While the MEPS for eukaryotes appears to be somewhat larger than that for prokaryotes, the lengths of homology required for intrachromosomal recombination between direct repeats in yeast and mammalian cells are quite similar, approximately 250 to 300 bp (18,19,38,39 (10,20,35,37), underscoring the importance of perfect, uninterrupted homology, and not just overall length of targeting homology, for effective targeted homologous recombination.…”

mentioning

confidence: 99%

Homology dependence of targeted recombination at the Chinese hamster APRT locus.

Scheerer¹,

Adair²

1994

Mol. Cell. Biol.

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Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.Gene targeting, targeted homologous recombination between an introduced DNA sequence and its endogenous chromosomal locus, is becoming increasingly useful as a means of genetic manipulation in mammalian cells as well as in yeast cells, permitting precise correction, disruption, replacement, or site-directed modification of virtually any gene or chromosomal locus for which a cloned sequence is available (8, 9, 27). However, much of our present understanding of the mechanisms of recombination is derived from studies in yeast cells; considerably less is known about recombination in mammalian cells. Most mammalian gene targeting studies have been directed toward disruption (knockout) or site-directed mutation of selected target gene loci in mouse embryo stem (ES) cells (8, 9), with their primary objective being to obtain the desired mutant mouse as quickly and easily as possible. Relatively few studies have examined targeted homologous recombination in cell types other than mouse ES cells.We have previously described a model system that has proven to be quite useful for studying targeted homologous recombination at the endogenous adenine phosphoribosyltransferase (APRT) locus in Chinese hamster ovary (CHO) cells (1-3). Our system utilizes a hemizygous, nonrevertible APRT deletion mutant (CHO-ATS49tg) as a recipient cell line, permitting direct selection of APRT+ recombinants arising by homologous recombination of APRT targeting sequences with the defective chromosomal gene locus. The 3-bp deletion in exon 5 of the CHO-ATS49tg APRT gene has eliminated an MboII restriction site, providing a useful restriction fragment length marker for Southern b...

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“…In this regard, Letsou and Liskay [27] have shown that the frequency of intrachromosomal gene conversion is sensitive to even a single base-pair mismatch within a 1-kb interval. Interestingly, Miller et al [28] have recently reported that a Ren-1 D targeting vactor homologously recombined only with the Ren-1 D locus of EK-CP1 ES cells which possess both the Ren-1D and Ren-2 genes. This is in spite of the fact that both renin loci are highly homologous.…”

Section: Discussionmentioning

confidence: 99%

Isolation of the Mouse Ren-1C Gene and Characterization of Renin Gene Expression in Both ES-D3 Cells and Their Parental Mouse Strain.

Tanimoto

Tamura

Sugiyama

et al. 1993

Journal of Reproduction and Development

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Renin is the rate-limiting enzyme in the renin-angiotensin system, the only known biochemical cascade that produces the potent octapeptide effector molecule angiotensin II that controls, to a large degree, cardiovascular homeostasis and reproductive function in mammals.In the present study, we have cloned the Ren-1C gene from the C57BL/6 mouse and determined its genomic structure.In order to provide the molecular basis for the application of the gene targeting method to the renin gene, we also characterized the structure and expression of the renin gene in embryonic stem D-3 (ES-D3) cells and in its parental mouse strains, 129/Sv mouse. The Ren-1C gene is 12 kilobases long and is composed of 9 exons interrupted by 8 introns. Renin mRNA was undetectable in ES-D3 cells but was found to be highly expressed in the submandibular gland of the 129/Sv mouse, in addition to the kidney. Southern blot analysis reveals that ES-D3 cells carry the additional duplicated renin gene, Ren-2, consistent with the classification of the 129/Sv mouse as a two-renin gene strain.

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Targeted integration of the Ren-1D locus in mouse embryonic stem cells.

Cited by 21 publications

References 33 publications

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Homology dependence of targeted recombination at the Chinese hamster APRT locus.

Isolation of the Mouse Ren-1C Gene and Characterization of Renin Gene Expression in Both ES-D3 Cells and Their Parental Mouse Strain.

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