Insertion of a foreign gene into the β-casein locus by Cre-mediated site-specific recombination

The expression of foreign genes in transgenic animals is generally unpredictable as transgenes are integrated at random after pro-nuclear injection into fertilized oocytes. In many cases, transgene expression is inhibited by neighbouring chromatin structures or by the repeated nature of the multiple transgene copies present at the integration site. A strategy involving homologous and site-specific recombination has been devised by which single copies of a foreign gene can be inserted specifically into the locus of a highly expressed gene. As a first step, a loxP recombination target site is introduced by homologous recombination into a predetermined gene locus such that the loxP sequence is placed next to the promoter region and replaces the translational initiation signal. In a subsequent site-specific recombination reaction, a gene of interest can be integrated into the pre-existing loxP site. This biphasic recombination strategy was used to integrate a luciferase reporter gene into the locus of the murine β-casein gene in embryonic stem cells.     

Position effect variegation and imprinting of transgenes in lymphocytes

Sequences proximal to transgene integration sites are able to deregulate transgene expression resulting in complex position effect phenotypes. In addition, transgenes integrated as repeated arrays are susceptible to repeat-induced gene silencing. Using a Cre recombinase-based system we have addressed the influence of transgene copy number (CN) on expression of hCD2 transgenes. CN reduction resulted in a decrease, increase or no effect on variegation depending upon the site of integration. This finding argues that repeat-induced gene silencing is not the principle cause of hCD2 transgene variegation. These results also suggest that having more transgene copies can be beneficial at some integration sites. The transgenic lines examined in this report also exhibited a form of imprinting, which was manifested by decreased levels of expression and increased levels of variegation, upon maternal transmission; and this correlated with DNA hypermethylation and a reduction in epigenetic chromatin modifications normally associated with active genes.     

BAC TG-EMBED: one-step method for high-level,copy-number-dependent,position-independent transgene expression

Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.     

Some characteristics of transgenic clones of mouse R1 line embryonic stem cells

Transgenic clones of mouse embryonic stem cells of the R1 line were received by transfection of plasmid linear vectors. The changes in the transgene structure during its integration into the genome of the target cells were investigated. Displacements were found on the flanks of the integrated transgene. It was found that multicopy tandem structures are formed in head–tail orientation at the transgene integration. It was noted that the number of copies of the integrated transgenes varies considerably, but the introduction of DNA fragments isolated from the nuclear envelopes into the flanks of the transgene normalizes the number of its copies.     

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.     

Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences,short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing

In genetically transformed plants, transgene silencing has been correlated with multiple and complex insertions of foreign DNA, e.g. T-DNA and vector backbone sequences. Occasionally, single-copy transgenes also suffer transgene silencing. We have compared integration patterns and T-DNA/plant DNA junctions in a collection of 37 single-copy T-DNA-transformed Arabidopsis lines, of which 13 displayed silencing. Vector sequences were found integrated in five lines, but only one of these displayed silencing. Truncated T-DNA copies, positioned in inverse orientation to an intact T-DNA copy, were discovered in three lines. The whole nptII gene with pnos promoter was present in the truncated copy of one such line in which heavy silencing has been observed. In the two other lines no silencing has been observed over five generations. Thus, vector sequences and short additional T-DNA sequences are not sufficient or necessary to induce transgene silencing. DNA methylation of selected restriction endonuclease sites could not be correlated with silencing. Our collection of T-DNA/plant DNA junctions has also been used to evaluate current models of T-DNA integration. Data for some of our lines are compatible with T-DNA integration in double-strand breaks, while for others initial invasion of plant DNA by the left or by the right T-DNA end seem important.     

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.