1,5-isoquinolinediol increases the frequency of gene targeting by homologous recombination in mouse fibroblasts

2003 ◽  
Vol 81 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Alexandre Semionov ◽  
Denis Cournoyer ◽  
Terry Y.-K Chow
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Dna Recombination ◽  
Illegitimate Recombination ◽  
Recombination Reaction ◽  
Absolute Frequency ◽  
Chromosomal Dna ◽  
The Absolute ◽  
Targeting Vector

Gene targeting is a technique that allows the introduction of predefined alterations into chromosomal DNA. It involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In theory, gene targeting constitutes the ideal method of gene therapy for single gene disorders. In practice, gene targeting remains extremely inefficient for at least two reasons: very low frequency of homologous recombination in mammalian cells and high proficiency of the mammalian cells to randomly integrate the targeting vector by illegitimate recombination. One known method to improve the efficiency of gene targeting is inhibition of poly(ADP-ribose)polymerase (PARP). It has been shown that PARP inhibitors, such as 3-methoxybenzamide, could lower illegitimate recombination, thus increasing the ratio of gene targeting to random integration. However, the above inhibitors were reported to decrease the absolute frequency of gene targeting. Here we show that treatment of mouse Ltk cells with 1,5-isoquinolinediol, a recent generation PARP inhibitor, leads to an increase up to 8-fold in the absolute frequency of gene targeting in the correction of the mutation at the stable integrated HSV tk gene.Key words: DNA recombination, gene targeting, PARP inhibition.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

scholarly journals Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4509-4517
Author(s):  
P Hasty ◽  
J Rivera-Pérez ◽  
C Chang ◽  
A Bradley
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Homologous Sequence ◽  
Reciprocal Recombination ◽  
Replacement Vector ◽  
Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

236 GENE TARGETING USING ZINC FINGER NUCLEASES (ZFN) IN THE PORCINE FIBROBLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 230
Author(s):  
S. Kim ◽  
J. W. Kim ◽  
S. M. Lee ◽  
J. H. Kim ◽  
M. J. Kang
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Zinc Finger ◽  
Marker Gene ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Domestic Animals ◽  
Zinc Finger Nucleases ◽  
Exogenous Dna ◽  
Targeting Vector

Gene targeting is a genetic technique that utilises homologous recombination between an engineered exogenous DNA fragment and the endogenous genome of an animal. In domestic animals, gene targeting has provided an important tool for producing knockout pigs for the α1,3-galactosyltransferase gene (GGTA1) to use in xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse embryonic stem cells. The application of gene targeting to somatic cells has been limited by its low efficiency. Recently, knockout rat and mouse were generated by introducing nonhomologus end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knockout and knock-in techniques in domestic animals is very important in biomedical research. In this study, we investigated homologous recombination events at the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. The CMAH-targeted ZFN plasmid and mRNA were purchased from Sigma-Aldrich (St Louis, MO, USA). Porcine ear fibroblasts cells were obtained from a 10-day-old male Chicago miniature pigs. The fibroblasts were cultured in DMEM containing 15% fetal bovine serum, 1 × nonessential amino acids, 1 × sodium pyruvate, 10–4 M β-mercaptoethanol, 100 unit mL–1 penicillin and 100 μg mL–1 streptomycin. The cells were trypsinized and resuspended at a concentration of 1.25 × 107 cells mL–1 in F10 nutrient mixture. Four hundred microliters of the cell suspension was electroporated in a 4-mm cuvette with 4 pulses of 1 ms duration using 400V capacitive discharges using the CMAH neo targeting vector and ZFN plasmid or RNA. The CMAH neo targeting vector consists of the neomycin resistance gene (neo) as a positive selectable marker gene, 789-bp 5′ arm and 763-bp 3′ arm from exon 8 of CMAH gene. After selection of G-418, PCR analysis was performed using 64 colonies transfected with ZFN plasmid and 48 colonies transfected with ZFN RNA. As a result, 19 positive colonies were identified in colonies transfected with ZFN plasmid and 15 colonies were identified in colonies transfected with ZFN RNA. The targeting efficiency was 29.7 and 31.6% in the colonies transfected with ZFN plasmid and ZFN RNA, respectively. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in production of CMAH knockouts for xenotransplantation.

scholarly journals Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

1997 ◽  
Vol 17 (1) ◽  
pp. 267-277 ◽  
Author(s):  
R G Sargent ◽  
M A Brenneman ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Repair Process ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Site Specific ◽  
Strand Breaks

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

scholarly journals Evidence for Biased Holliday Junction Cleavage and Mismatch Repair Directed by Junction Cuts during Double-Strand-Break Repair in Mammalian Cells

2001 ◽  
Vol 21 (10) ◽  
pp. 3425-3435 ◽  
Author(s):  
Mark D. Baker ◽  
Erin C. Birmingham
Keyword(s):  
Homologous Recombination ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Holliday Junction ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Break Repair

ABSTRACT In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.

scholarly journals Processing of DNA prior to illegitimate recombination in mouse cells.

1997 ◽  
Vol 17 (7) ◽  
pp. 3779-3785 ◽  
Author(s):  
G Henderson ◽  
J P Simons
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Illegitimate Recombination ◽  
Strand Bias ◽  
Dna Breaks ◽  
Exogenous Dna ◽  
Double Strand Dna Breaks ◽  
Mouse Cells ◽  
Exonuclease Digestion

In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 5'-->3' exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 3'-->5' exonuclease processing also occurs.

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