Complex patterns of inheritance of an imprinted murine transgene suggest incomplete germline erasure

Here we report a transgenic mouse line that exhibits significant deviations from a classic pattern of parental imprinting. When the transgene is passed through the female germline, it is completely silenced in some offspring while in others expression is reduced. This variable expressivity does not appear to be the result of differences in the presence of unlinked modifiers. Female transmission of the transgene is associated with hypermethylation. The transgene is generally reactivated on passage through the male germline. Extended pedigrees reveal complex patterns of inheritance of the phenotype. The most likely explanation for this result is that the imprint is not completely erased and reset when passed through the germline of either sex. FISH analysis reveals that the transgene has integrated into chromosome 3 band E3, a region not known to carry imprinted genes, and the integration site shows no sign of allele-specific differential methylation. These findings, in conjunction with other recent work, raise the possibility that the introduction of foreign DNA into the mammalian genome, either through retrotransposition or transgenesis, may be associated with parental imprinting that is not always erased and reset during meiosis.     

Sensitive flow cytometric analysis reveals a novel type of parent-of-origin effect in the mouse genome

The discovery of classic parental imprinting came, at least in part, from the analysis of transgene expression in mice. It was noticed that some transgenes were only expressed following paternal transmission and that others sometimes showed differential patterns of methylation depending on the parent of origin. Here, we present evidence of a novel and more subtle form of parental imprinting by taking advantage of the highly sensitive detection of murine transgene expression afforded by flow cytometry. We have produced nine lines of transgenic mice carrying a GFP reporter linked to the human alpha-globin promoter and enhancer elements, which direct expression to erythroid cells. A high proportion of transgenic lines, four of the nine, display significantly lower levels of expression following maternal transmission. Both the percentage of expressing cells and the mean fluorescence in expressing cells are between 10% and 30% lower following maternal transmission. These effects are reversible upon passage through the opposite germline. This finding raises the possibility that differences in the epigenetic state of the maternal and paternal chromosomes in adult somatic cells are more widespread than was previously thought.     

Distinct Requirements for Somatic and Germline Expression of a Generally Expressed Caernorhabditis Elegans Gene

In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germline expression of repetitive sequences.     

Disruption of the bipartite imprinting center in a family with Angelman syndrome

Imprinting in 15q11-q13 is controlled by a bipartite imprinting center (IC), which maps to the SNURF-SNRPN locus. Deletions of the exon 1 region impair the establishment or maintenance of the paternal imprint and can cause Prader-Willi syndrome (PWS). Deletions of a region 35 kb upstream of exon 1 impair maternal imprinting and can cause Angelman syndrome (AS). So far, in all affected sibs with an imprinting defect, an inherited IC deletion was identified. We report on two sibs with AS who do not have an IC deletion but instead have a 1-1.5 Mb inversion separating the two IC elements. The inversion is transmitted silently through the male germline but impairs maternal imprinting after transmission through the female germline. Our findings suggest that the close proximity and/or the correct orientation of the two IC elements are/is necessary for the establishment of a maternal imprint.     

A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development

A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid‐gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re‐established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely‐flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non‐CpG cytosines of the retroelement promoter were highly methylated especially in the transgene‐active mid‐gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid‐gestational embryo.     

Proper expression of myosin genes in transgenic nematodes.

Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product.     

Investigation of Elements Sufficient To Imprint the Mouse Air Promoter

Imprinted maternal-allele-specific expression of the mouse insulin-like growth-factor type 2 receptor (Igf2r) gene depends on a 3.7-kb element named region 2, located in the second intron of the gene. Region 2 carries a maternal-allele-specific methylation imprint and contains an imprinted CpG island promoter (Air) that expresses a noncoding antisense RNA from the paternal inherited allele only. Here, we use transgenes to test the minimal requirements for imprinting of Air and to test if the action of region 2 is restricted to Igf2r. Transgenes up to 9 kb with Air as a single promoter are expressed but not imprinted. When coupled to the Igf2r CpG island promoter on a 44-kb transgene, Air was imprinted in one of three lines. However, Air on a 4.6-kb fragment is also imprinted in 2 of 14 lines when inserted in an intron of an adenine phosphoribosyltransferase (Aprt) transgene, and in one line, the imprinted methylation and expression of Air have been transferred onto the Aprt CpG island promoter. These data suggest that a dual CpG island promoter setting may facilitate Air imprinting as a short transgene and also show that Air can transfer imprinting onto other genes. However, for reliable Air imprinting, elements are necessary that are located outside a 44-kb region spanning the Air-Igf2r promoters.     

Gtl2 lacZ,an insertional mutation on mouse Chromosome 12 with parental origin-dependent phenotype

We have produced a transgenic mouse line, Gtl2 ^lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2 ^lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2 ^lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2 ^lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2 ^lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed. Received: 30 May 1995 / Accepted: 7 August 1995     

Successful transgenesis of the parasitic nematode Strongyloides stercoralis requires endogenous non-coding control elements

Critical investigations into the cellular and molecular biology of parasitic nematodes have been hindered by a lack of modern molecular genetic techniques for these organisms. One such technique is transgenesis. To our knowledge, the findings reported here demonstrate the first heritable DNA transformation and transgene expression in the intestinal parasite Strongyloides stercoralis. When microinjected into the syncitial gonads of free-living S. stercoralis females, a construct fusing the S. stercoralis era-1 promoter, the coding region for green fluorescent protein (gfp) and the S. stercoralis era-1 3' untranslated region was expressed in intestinal cells of normally developing F1 transgenic larvae. The frequency of transformation and GFP expression among F1 larvae was 5.3%. By contrast, expression of several promoter::gfp fusions incorporating only Caenorhabditis elegans regulatory elements was restricted to abortively developing F1 embryos of S. stercoralis. Despite its lack of regulated expression, PCR revealed that one of these C. elegans-based vector constructs, the sur-5::gfp fusion, is incorporated into F1 larval progeny of microinjected female worms and then transmitted to the F2 through F5 generations during two host passages conducted without selection and punctuated by free-living generations reared in culture. Heritable DNA transformation and regulated transgene expression, as demonstrated here for S. stercoralis, constitute the essential components of a practical system for transgenesis in this parasite. This system has the potential to significantly advance the molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally.     

Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.     

Specific differentially methylated domain sequences direct the maintenance of methylation at imprinted genes

Landmark features of imprinted genes are differentially methylated domains (DMDs), in which one parental allele is methylated on CpG dinucleotides and the opposite allele is unmethylated. Genetic experiments in the mouse have shown that DMDs are required for the parent-specific expression of linked clusters of imprinted genes. To understand the mechanism whereby the differential methylation is established and maintained, we analyzed a series of transgenes containing DMD sequences and showed that imperfect tandem repeats from DMDs associated with the Snurf/Snrpn, Kcnq1, and Igf2r gene clusters govern transgene imprinting. For the Igf2r DMD the minimal imprinting signal is two unit copies of the tandem repeat. This imprinted transgene behaves identically to endogenous imprinted genes in Dnmt1o and Dnmt3L mutant mouse backgrounds. The primary function of the imprinting signal within the transgene DMD is to maintain, during embryogenesis and a critical period of genomic reprogramming, parent-specific DNA methylation states established in the germ line. This work advances our understanding of the imprinting mechanism by defining a genomic signal that dependably perpetuates an epigenetic state during postzygotic development.     

Creation of low-copy integrated transgenic lines in Caenorhabditis elegans

In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing.     

Epigenetic control of transgene expression and imprinting by genotype-specific modifiers

Expression and DNA methylation of the transgene locus TKZ751 are controlled by genotype-specific modifier genes. The DBA/2 and 129 genetic backgrounds enhanced expression, while the BALB/c background suppressed expression, but only following maternal inheritance of the BALB/c modifier. Epigenetic modification of the transgene locus was cumulative over successive generations, which in BALB/c mice resulted in an irreversible methylation after three consecutive germline passages. Therefore, at the TKZ751 locus the germline fails to reverse previously acquired epigenetic modifications, a process that is usually essential to restore the genomic totipotency. Hence the genotype-specific modifier genes regulate penetrance and expressivity as well as parental imprinting of the TKZ751 locus through epigenetic modification.     

Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus

Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.     

Efficient production of germline transgenic chickens using lentiviral vectors

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.     

Heritable Loss of Replication Control of a Minichromosome Derived from the B Chromosome of Maize

During an accumulation regime of a small telomere-truncated B chromosome, a derivative with large variations in size and multiple punctate centromere loci exhibiting amplified copy numbers was discovered. Multiple centromere satellite loci or transgene signals were documented in amplified chromosomes, suggesting over-replication. Immunolocalization studies revealed multiple foci of biochemical markers characteristic of active centromeres such as CENP-C and phosphorylation of histones H3S10 and H2AThr133. The amplified chromosomes exhibit an absence of chromosome disjunction in meiosis I and an infrequent chromosome disjunction in meiosis II. Despite their unusual structure and behavior these chromosomes were observed in the lineage for seven generations during the course of this study. While severely truncated relative to a normal B chromosome, the progenitor minichromosome is estimated to be at least several megabases in size. Given that the centromere and transgene signals at opposite ends of the chromosome generally match in copy number, the replication control is apparently lost over several megabases.     

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.     

Developmental consequences of imprinting of parental chromosomes by DNA methylation

Genomic imprinting by epigenetic modifications, such as DNA methylation, confers functional differences on parental chromosomes during development so that neither the male nor the female genome is by itself totipotential. We propose that maternal chromosomes are needed at the time when embryonic cells are totipotential or pluripotential, but paternal chromosomes are probably required for the proliferation of progenitor cells of differentiated tissues. Selective elimination or proliferation of embryonic cells may occur if there is an imbalance in the parental origin of some alleles. The inheritance of repressed and derepressed chromatin structures probably constitutes the initial germ-line-dependent 'imprints'. The subsequent modifications, such as changes in DNA methylation during early development, will be affected by the initial inheritance of epigenetic modifications and by the genotype-specific modifier genes. A significant number of transgene inserts are prone to reversible methylation imprinting so that paternally transmitted transgenes are undermethylated, whereas maternal transmission results in hypermethylation. Hence, allelic differences in epigenetic modifications can affect their potential for expression. The germ line evidently reverses the previously acquired epigenetic modifications before the introduction of new modifications. Errors in the reversal process could result in the transmission of epigenetic modifications to subsequent generation(s) with consequent cumulative phenotypic and grandparental effects.