Identifying and genotyping transgene integration loci

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.     

动物基因组定点整合转基因技术研究进展

传统转基因技术,如显微注射、转座子、慢病毒转染等将目的基因插入基因组内的整合方式是随机的,这些随机整合对后期转基因动物品系组建和育种带来诸多不利,因此有研究人员提出了定点整合转基因技术。目前该技术的定点整合效率非常低,主要取决于两个方面：一是靶位点产生DNA双链断裂(double-strand break, DSB)的效率;二是断裂后的靶位点与携带同源臂及外源基因的供体质粒发生同源重组的效率,其中同源重组修复(homologous recombination repair, HDR)是基因组定点整合最为依赖的修复机制。靶位点产生DSB后,机体的DNA修复既可能发生HDR,也可能发生非同源末端连接(nonhomologous end joining, NHEJ),并且两者之间存在竞争关系,因此激活HDR或抑制NEHJ都可提高定点整合转基因的效率。本文结合影响定点整合的因素,对提高定点整合效率最新探索方面进行了综述。     

Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice.

In line 4 transgenic mice, six to eight copies of a 50-kb lambda recombinant clone are arranged in a head-to-tail tandem array on chromosome 3. Embryos homozygous for the transgene become arrested in their development on Day 5 of gestation shortly after implantation. The insertion site was cloned using a small segment of the transgene as a probe. Comparison of the insertion site with the wildtype locus indicates that a 22-kb deletion of host DNA has occurred in line 4 mice. Restriction enzyme analyses showed that neither the tandem array nor the flanking chromosomal DNA had any detectable rearrangements. Sequencing of the junctions between host and foreign DNA, however, revealed insertions of small fragments of DNA of unknown origin as well as an insertion of a DNA segment derived from another region of the transgene. Therefore, disruption of an essential gene in the line 4 transgenic mouse may have been caused by the insertion of 300-400 kb of foreign DNA or a deletion of sequences in the host genome.     

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.     

Structural features of the integration site of foreign DNA in the transgenic mouse genome

The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome.     

A human t-PA mutant cDNA cassette knocked in the murine fgfr-4 locus targeting for mammary gland expression

The concept of using animal mammary glands asbioreactors to produce recombinant pharmaceuticalproteins has been widely accepted for great potentialcommercial interests [1]. Up to now, the main method tomake transgenic animals is microinjection [2,3]. Lowlevel and unpredictability of the foreign gene expressionwere found among transgenic lines. The major reason isthat the microinjected foreign gene is integrated into thegenome randomly as a stretch of multiple copies, and thesurrounding chromat…     

Spontaneous germline amplification and translocation of a transgene array.

The majority of the mammalian genome is thought to be relatively stable throughout and between generations. There are no developmentally programmed gene amplifications as seen in lower eukaryotes and prokaryotes, however a number of unscheduled gene amplifications have been documented. Apart from expansion of trinucleotide repeats and minisatellite DNA, which involve small DNA elements, other cases of gene or DNA amplifications in mammalian systems have been reported in tumor samples or permanent cell lines. The mechanisms underlying these amplifications remain unknown. Here, we report a spontaneous transgene amplification through the male germline which resulted in silencing of transgene expression. During routine screening one mouse, phenotypically negative for transgene expression, was found to have a transgene copy number much greater than that of the transgenic parent. Analysis of the transgene expansion revealed that the amplification in the new high copy transgenic line resulted in a copy number approximately 40-60 times the primary transgenic line copy number of 5-8 copies per haploid genome. Genetic breeding analysis suggested that this amplification was the result of insertion at only one integration site, that it was stable for at least two generations and that the site of insertion was different from the site at which the original 5-8 copy array had integrated. FISH analysis revealed that the new high copy array was on chromosome 7 F3/4 whereas the original low copy transgene array had been localised to chromosome 3E3. DNA methylation analysis revealed that the high copy transgene array was heavily methylated. The amplification of transgenes, although a rare event, may give insight into amplification of endogenous genes which can be associated with human disease.     

Inherited haemolytic anaemia created by insertional inactivation of the α-spectrin gene

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.     

A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

Identification of the Genomic Insertion Site of Pmel-1 TCR α and β Transgenes by Next-Generation Sequencing

The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8⁺ T cell differentiation, autoimmunity and adoptive immunotherapy. The ‘zygosity’ of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively ‘shallow’ (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.     

The distribution of transgene insertion sites in barley determined by physical and genetic mapping

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.     

Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity

When transgenic mice are created by microinjection of DNA into the pronucleus, the sites of DNA integration into the mouse genome cannot be predicted. Most methods based on polymerase chain reaction (PCR) that have been used for determining the integration site of foreign DNA into a genome require specific reagents and/or complicated manipulations making routine use tedious. In this report we demonstrate the use of a PCR-based method-TAIL-PCR (Thermal Asymmetric Interlaced PCR) which relies on a series of PCR amplifications with gene specific and degenerate primers to reliably amplify the integration sites. By way of example, using this approach, three separate integration sites were found (on chromosomes 8, 15 and 17) in one transgenic founder. As the sites on chromosomes 8 and 15 failed to segregate in any subsequent progeny, whole chromosome paints were done to determine if translocations involving chromosomes 8 and 15 occurred at the time of transgene integration. Whole chromosome painting could not detect translocations, suggesting that the rearrangements likely involve only small stretches of chromosomes. Site-specific primers were used to identify the progeny carrying only one integration site; these mice were then used as sub-founders for subsequent breedings. Integration site specific primers were used to distinguish homozygous progeny from heterozygotes. TAIL-PCR thus provides an easy and reliable way to (1) identify multiple integration sites in transgenic founders, (2) select breeders with one integration site, and (3) determine zygosity in subsequent progeny. Use of this strategy may also be considered to map integration sites in situations of unexpected phenotype or embryonic lethality while creating new transgenic mice.     

Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA

Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.     

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.     

Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking.

Transgenes can affect transgenic mice via transgene expression or via the so-called positional effect. DNA sequences can be localized in chromosomes using recently established mouse genomic databases. In this study, we describe a chromosomal mapping method that uses the genomic walking technique to analyze genomic sequences that flank transgenes, in combination with mouse genome database searches. Genomic DNA was collected from two transgenic mouse lines harboring pCAGGS-based transgenes, and adaptor-ligated, enzyme restricted genomic libraries for each mouse line were constructed. Flanking sequences were determined by sequencing amplicons obtained by PCR amplification of genomic libraries with transgene-specific and adaptor primers. The insertion positions of the transgenes were located by BLAST searches of the Ensembl genome database using the flanking sequences of the transgenes, and the transgenes of the two transgenic mouse lines were mapped onto chromosomes 11 and 3. In addition, flanking sequence information was used to construct flanking primers for a zygosity check. The zygosity (homozygous transgenic, hemizygous transgenic and non-transgenic) of animals could be identified by differential band formation in PCR analyses with the flanking primers. These methods should prove useful for genetic quality control of transgenic animals, even though the mode of transgene integration and the specificity of flanking sequences needs to be taken into account.     

Characterization of Growth and Reproduction Performance,Transgene Integration,Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.     

Identification of the transgenic integration site in 2C T cell receptor transgenic mice

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vβ13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3′ region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.     

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.