Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens

In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates predominantly by homologous recombination with its genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). Overall homology of only 1 kb is sufficient to achieve a 50% yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement following two homologous recombination events. Allele replacement frequency depends on the symmetry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the transforming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single insertions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20% of all transformants when the minimum requirements for allele replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants.     

The length of homology required for gene targeting in embryonic stem cells.

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.     

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

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.     

The effects of terminal heterologies on gene targeting by insertion vectors in embryonic stem cells.

We have examined the effects of placing nonhomologous DNA on the ends of an insertion-type gene targeting vector. The presence of terminal heterologies was found to be compatible with insertion targeting, and the terminal heterologies were efficiently removed. Terminal heterologies reduced the frequency of gene targeting to variable extents. The degree of inhibition of targeting was dependent on the length and the position of the heterology: 2.1kb heterologous sequences were more inhibitory than shorter regions of heterology, and heterology placed on the end of the long (4.8kb) arm of homology was more inhibitory than heterology positioned on the end of the short (0.8kb) arm. When heterology was placed on both arms of the targeting vector the targeting efficiencies were similar to or higher than when heterology was present on the long arm only. These results suggest that terminal sequences are removed simultaneously from both ends of targeting vectors. The removal of terminal sequences probably occurs by exonucleolytic degradation of both strands at each end, and removal of at least one of the strands is intimately coupled with the process of homologous recombination. These findings have implications for the design of gene targeting vectors.     

Genetic stability of gene-targeted immunoglobulin loci. II. Influence of the cell line and the vector linearization site

The site-specific integration of exogenous gene fragments by homologous recombination provides a convenient method for altering the immunoglobulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybridoma cells we compared the recombination frequencies of vectors that could be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombination was observed, irrespective of the location of the site at which the vector was cleaved. Integration events involving the human IgG1 vectors were lost at high frequency due to secondary vector excision, so that all stable recombinations were found to be replacement events. Replacement recombination of an integration vector involves an illegitimate crossover at least at the 3′ side and sometimes gives rise to deletion of the C_H1 domain. However, a homologous event at the 3′ side is more efficient than an illegitimate one, so that a homology that is distributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total length. The position of the linearization site in the vector markedly influenced the targeting efficiency, but surprisingly, whether a double-strand break in the homology or in the heterology region more efficiently promoted integration was dependent on the cell line. In all cells, however, cleavage of the vector outside the homology region favoured stable replacements with a bias against C_H1-truncated clones. We further show that the frequency of replacements induced by integration vectors is not correlated to the homology length and cannot be increased by irradiation of the cells. Our findings indicate that for targeting the IgH locus other mechanisms might be involved than at other loci. Received: 20 January 1997 / Accepted: 9 June 1997     

Genetic stability of gene-targeted immunoglobulin loci. II. Influence of the cell line and the vector linearization site

The site-specific integration of exogenous gene fragments by homologous recombination provides a convenient method for altering the immunoglobulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybridoma cells we compared the recombination frequencies of vectors that could be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombination was observed, irrespective of the location of the site at which the vector was cleaved. Integration events involving the human IgG1 vectors were lost at high frequency due to secondary vector excision, so that all stable recombinations were found to be replacement events. Replacement recombination of an integration vector involves an illegitimate crossover at least at the 3′ side and sometimes gives rise to deletion of the C_H1 domain. However, a homologous event at the 3′ side is more efficient than an illegitimate one, so that a homology that is distributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total length. The position of the linearization site in the vector markedly influenced the targeting efficiency, but surprisingly, whether a double-strand break in the homology or in the heterology region more efficiently promoted integration was dependent on the cell line. In all cells, however, cleavage of the vector outside the homology region favoured stable replacements with a bias against C_H1-truncated clones. We further show that the frequency of replacements induced by integration vectors is not correlated to the homology length and cannot be increased by irradiation of the cells. Our findings indicate that for targeting the IgH locus other mechanisms might be involved than at other loci.     

Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania.

In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania. We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome. We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency. No homologous recombination was detected, however, when the flanking homologous regions were <180 bp. A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches. Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles. Different chromosomal locations were found to be targeted with significantly variable levels of efficiency. Finally, different strains of the same species showed differences in gene targeting frequency. Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.     

The effects of polymorphisms on human gene targeting

DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.     

Myostatin基因打靶载体的构建及猪胎儿成纤维细胞Myostatin基因的敲除

目的:用缺口修复等技术构建Myostatin(肌肉生长抑制素,MSTN)基因打靶载体,并对大白猪胎儿成纤维体细胞进行转染,获得基因敲除细胞。方法与结果:首先构建用于MSTN基因同源长臂(LA)的抓捕载体,然后在大肠杆菌内利用Red同源重组系统介导的缺口修复,从含大白猪MSTN基因座的细菌人工染色体上亚克隆9.9 kb的LA到抓捕载体上,经过部分序列测定,同源性为100%;通过PCR获得1.4 kb的同源短臂(SA);将LA和SA连入载体pLOXP,构建含有neo和tk正负筛选标记基因的MSTN基因打靶载体pLOXP-MSTN-KO;将线性化的pLOXP-MSTN-KO通过电转染整合到大白猪胎儿成纤维细胞基因组中,利用G418和丙氧鸟苷进行药物筛选,获得抗性细胞克隆890个,通过PCR和DNA测序鉴定获得基因敲除的细胞克隆4个。结论:构建了有效的MSTN基因打靶载体,通过转染获得基因敲除细胞,为利用体细胞核移植制备MSTN基因敲除猪奠定了基础。     

Sequence Analysis of a 685-kb Genomic Region on Chromosome 3p22-p21.3 That Is Homozygously Deleted in a Lung Carcinoma Cell Line

Frequent chromosomal aberrations and/or losses of heterozygosityinvolving the short arm of chromosome 3 in carcinomas of thelung, kidney and other tissues imply that multiple putativetumor suppressor genes may be present on this chromosomal arm.To search for one of these genes, we determined DNA sequencesin the genomic region at 3p22–21.3 where we had previouslydetected a homozygous deletion in a lung cancer cell line. TheDNA sequence results of an about 685-kb region indicated thatthe size of the homozygously deleted segment was 638,489 bp,in which we identified only four genes including the integrinRLC and the trans-Golgi p230 genes, both reported previously.The predicted amino acid sequences of one of the two novel genesshowed high homology to villin, a human cytoskeleton protein;those of the other gene, termed HYA22, revealed significanthomology to YA22, a hypothetical protein predicted from DNAsequences of Schizosaccharomyces pombe. The computer programsHEXON or GRAIL were able to predict three-fourths of the exons;the smallest exon predicted by either program was 46 base pairs.Repetitive sequences contained in the genomic region included151 copies of the Alu sequence (1 copy/every 4.5 kb), 19 copiesof the L1 sequence (1 copy/every 36 kb) , and 10 copies of theTHE sequence.     

Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes

The generation of specific mutant animal models is critical for functional analysis of human genes. The conventional gene targeting approach in embryonic stem cells (ESCs) by homologous recombination is however laborious, slow, expensive, and limited to species with functional ESCs. It is therefore a long-sought goal to develop an efficient and simple alternative gene targeting strategy. Here we demonstrate that, by combining an efficient ZFN pair and ssODN, a restriction site and a loxP site were successfully introduced into a specific genomic locus. A targeting efficiency up to 22.22% was achieved by coinciding the insertion site and the ZFN cleavage site isogenic and keeping the length of the homology arms equal and isogenic to the endogenous target locus. Furthermore, we determine that ZFN and ssODN-assisted HR is ssODN homology arm length dependent. We further show that mutant alleles generated by ZFN and ssODN-assisted HR can be transmitted through the germline successfully. This study establishes an efficient gene targeting strategy by ZFN and ssODN-assisted HR in mouse zygotes, and provides a potential avenue for genome engineering in animal species without functional ES cell lines.     

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

Here we report an approach to generate a knock-in mouse model using an ‘ends-out’ gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed ‘ectopic gene targeting’ has so far only been described for ‘ends-in’ integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insertion-Duplication Mutagenesis in Streptococcus pneumoniae: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool

To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.     

Homozygous deletion of six olfactory receptor genes in a subset of individuals with Beta-thalassemia

Progress in the functional studies of human olfactory receptors has been largely hampered by the lack of a reliable experimental model system. Although transgenic approaches in mice could characterize the function of individual olfactory receptors, the presence of over 300 functional genes in the human genome becomes a daunting task. Thus, the characterization of individuals with a genetic susceptibility to altered olfaction coupled with the absence of particular olfactory receptor genes will allow phenotype/genotype correlations and vindicate the function of specific olfactory receptors with their cognate ligands. We characterized a 118 kb β-globin deletion and found that its 3' end breakpoint extends to the neighboring olfactory receptor region downstream of the β-globin gene cluster. This deletion encompasses six contiguous olfactory receptor genes (OR51V1, OR52Z1, OR51A1P, OR52A1, OR52A5, and OR52A4) all of which are expressed in the brain. Topology analysis of the encoded proteins from these olfactory receptor genes revealed that OR52Z1, OR52A1, OR52A5, and OR52A4 are predicted to be functional receptors as they display integral characteristics of G-proteins coupled receptors. Individuals homozygous for the 118 kb β-globin deletion are afflicted with β-thalassemia due to a homozygous deletion of the β-globin gene and have no alleles for the above mentioned olfactory receptors genes. This is the first example of a homozygous deletion of olfactory receptor genes in human. Although altered olfaction remains to be ascertained in these individuals, such a study can be carried out in β-thalassemia patients from Malaysia, Indonesia and the Philippines where this mutation is common. Furthermore, OR52A1 contains a γ-globin enhancer, which was previously shown to confer continuous expression of the fetal γ-globin genes. Thus, the hypothesis that β-thalassemia individuals, who are homozygous for the 118 kb deletion, may also have an exacerbation of their anemia due to the deletion of two copies of the γ-globin enhancer element is worthy of consideration.