“Parahomologous” Gene Targeting in Drosophila Cells: An Efficient, Homology-Dependent Pathway of Illegitimate Recombination Near a Target Site

Gene targeting is an in situ manipulation of an endogenous gene in a precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. Conventional gene targeting has been achieved through the use of embryonic stem cells. However, such a procedure is often long, tedious, and expensive and has been limited in the mouse only due to a lack of usable embryonic stem cells in other species. This study was carried out to develop a much simplified procedure of gene targeting using E. coli recombinase recA and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene. The single-stranded oligonucleotide to target exon 3 of HPRT was 74 bases in length and included three phosphorothioate linkages at each terminus (also known as S-oligo) so as to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except for a single nucleotide that induces a mismatch between the introduced oligonucleotide and endogenous HPRT gene. Although the exact mechanism is yet unknown, endogenous repairing of such a mismatch would give rise to the conversion of TAT to TAG stop codon, thereby losing the function of the target gene. Prior to an introduction into zygotes, modified single-stranded oligonucleotides were preincubated with recA recombinase to enhance the homologous recombination. The recA-oligonucleotide complex was microinjected into the pronuclei of zygotes. Individual microinjected embryos that developed to the blastocyst stage were analyzed for the expected nucleotide conversion using PCR and subsequent sequencing. The conversion of TAT to TAG stop codon was confirmed in two embryos among forty tested blastocysts, so that the frequency of gene targeting was approximately 5%. The result suggests that the gene targeting was feasible by this relatively easier direct method. Subsequent transfer of gene-targeted embryos to recipients to obtain transgenic mice missing the function of HPRT gene is underway. Further technical refinement and enhancement of homologous recombination frequency will be required for the practical use of this new approach for gene targeting in mice.

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

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab236 ◽

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.

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Processing of DNA prior to illegitimate recombination in mouse cells.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3779 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3779-3785 ◽

Cited By ~ 14

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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Homologous and illegitimate recombination in developing Xenopus oocytes and eggs

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6897-6906.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6897-6906

Author(s):

C W Lehman ◽

M Clemens ◽

D K Worthylake ◽

J K Trautman ◽

D Carroll

Keyword(s):

Homologous Recombination ◽

Developmental Stages ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Xenopus Laevis Oocytes ◽

Recombination Activity ◽

Exogenous Dna ◽

Strand Breaks ◽

Nuclear Extracts ◽

Reaction Proceeds

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.

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326 CONSTRUCTION OF A SPLICING-DEPENDENT SELECTABLE MARKER FOR GENE TARGETTING

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab326 ◽

2011 ◽

Vol 23 (1) ◽

pp. 259

Author(s):

S. Cernea ◽

K. Wells

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Splice Site ◽

Mammalian Cells ◽

Expression Vector ◽

Selectable Marker ◽

Stop Codon ◽

Intron Splice ◽

G418 Resistance ◽

Control Plasmid

Gene targeting in mammalian cells plays a crucial role in biotechnology. These experiments are characterised by low rates of homologous recombination and high rates of random integration. Therefore, many fibroblast colonies must be screened to identify a targeting event. To dramatically reduce the survival of random integration events, we have developed a splicing-dependent selectable marker strategy by introducing a mutation in a codon-optimized G418 resistance gene (mNeo). This mutation could be corrected upon homologous recombination. Since the C-terminal region of aminoglycoside phosphotransferase (AphII, Neo/Kan resistance) participates in formation of the active site of this enzyme, we hypothesised that addition of even one amino acid at the C-terminus would render this protein non-functional. To test this hypothesis, a mutation was introduced in an E. coli AphII expression vector that converted the stop codon of AphII to tryptophan (X265W, TGA > TGGTAA). This mutation was confirmed to inactivate AphII by independently characterising the G418 and Kanamycin resistance (or lack thereof) provided by the X265W mutation. To evaluate this mutation in mammalian cells, two intronless mammalian expression vectors were constructed that differed by the presence or absence of the X265W mutation. G418 resistance was only provided by the wildtype sequence, thus confirming that X265W inactivates AphII in mammalian cells. An identical mutation was then introduced into a eukaryotic expression vector based on mNEO. Further, the sequence was extended to create a 5′ intron splice site (TGA > TGGTAAGAGTT). This region was designed to direct splicing between the first and second G residues thus removing the G in the third position of the W codon. The 3′ intron splice sites was then designed to provide an A residue as the first base of the next exon so that successful splicing would correct the mutation by recreating an appropriately positioned stop codon (TGA). To evaluate this strategy in mammalian cells, two plasmids were constructed that harbored the X265W mutation embedded at the 5′ splice site of a downstream intron. In one plasmid (pSC3-G) the first base of the downstream exon begins with a G residue resulting in inactivation of AphII. In the other plasmid (pSC2-A), the first base of the downstream exon begisn with an A residue forming a stop codon that allows for active, wildtype AphII. These plasmids were transfected into porcine fetal fibroblasts and subjected to selection with G418. A positive control plasmid and pSC2-A produced colonies that were too numerous to count. A negative control plasmid and pSC3-G produced no colonies. It can be concluded that the X265W mutation can be corrected by splicing to an exon that begins with an A residue. This splicing-dependent selectable marker may prove useful in gene targeting experiments when the site of modification is followed by an exon that begins with an A.

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

Biochemistry and Cell Biology ◽

10.1139/o02-172 ◽

2003 ◽

Vol 81 (1) ◽

pp. 17-24 ◽

Cited By ~ 6

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.

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Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.93 ◽

1998 ◽

Vol 18 (1) ◽

pp. 93-101 ◽

Cited By ~ 212

Author(s):

Beth Elliott ◽

Christine Richardson ◽

Jamie Winderbaum ◽

Jac A. Nickoloff ◽

Maria Jasin

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Gene Targeting ◽

Mammalian Cells ◽

Embryonic Stem ◽

Sequence Divergence ◽

Recombinational Repair ◽

Exogenous Dna ◽

Extensive Degradation ◽

Recombination Gene

ABSTRACT Mammalian cells are able to repair chromosomal double-strand breaks (DSBs) both by homologous recombination and by mechanisms that require little or no homology. Although spontaneous homologous recombination is rare, DSBs will stimulate recombination by 2 to 3 orders of magnitude when homology is provided either from exogenous DNA in gene-targeting experiments or from a repeated chromosomal sequence. Using a gene-targeting assay in mouse embryonic stem cells, we now investigate the effect of heterology on recombinational repair of DSBs. Cells were cotransfected with an endonuclease expression plasmid to induce chromosomal DSBs and with substrates containing up to 1.2% heterology from which to repair the DSBs. We find that heterology decreases the efficiency of recombinational repair, with 1.2% sequence divergence resulting in an approximately sixfold reduction in recombination. Gene conversion tract lengths were examined in 80 recombinants. Relatively short gene conversion tracts were observed, with 80% of the recombinants having tracts of 58 bp or less. These results suggest that chromosome ends in mammalian cells are generally protected from extensive degradation prior to recombination. Gene conversion tracts that were long (up to 511 bp) were continuous, i.e., they contained an uninterrupted incorporation of the silent mutations. This continuity suggests that these long tracts arose from extensive degradation of the ends or from formation of heteroduplex DNA which is corrected with a strong bias in the direction of the unbroken strand.

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Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6897 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6897-6906 ◽

Cited By ~ 27

Author(s):

C W Lehman ◽

M Clemens ◽

D K Worthylake ◽

J K Trautman ◽

D Carroll

Keyword(s):

Homologous Recombination ◽

Developmental Stages ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Xenopus Laevis Oocytes ◽

Recombination Activity ◽

Exogenous Dna ◽

Strand Breaks ◽

Nuclear Extracts ◽

Reaction Proceeds

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.

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Gene Targeting

10.1093/oso/9780199637928.001.0001 ◽

1999 ◽

Cited By ~ 2

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.

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Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

Plant and Cell Physiology ◽

10.1093/pcp/pcab034 ◽

2021 ◽

Author(s):

Daisuke Miki ◽

Rui Wang ◽

Jing Li ◽

Dali Kong ◽

Lei Zhang ◽

...

Keyword(s):

Gene Targeting ◽

Agronomic Traits ◽

Higher Plants ◽

Crop Improvement ◽

Strand Break ◽

End Joining ◽

Homology Directed Repair ◽

Target Dna ◽

Targeted Gene Editing ◽

Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

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