Concatenation of Transgenic DNA: Random or Orchestrated?

Abstract

Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.

Characterizing a Lethal CAG-ACE2 Transgenic Mouse Model for SARS-CoV-2 infection with Using Cas9-Enhanced Nanopore Sequencing

“…Line #5 had 140 ± 27 copies in the genome, #6 - 72 ± 8, #10 - 73 ± 7. Inside the concatemer most copies are usually oriented in a head-to-tail direction, but palindromic junctions and truncated transgene copies could also be present (Smirnov and Battulin 2021). However, due to its repeated nature, the internal concatemer structure is extremely difficult to study.…”

The human EF1a promoter does not provide expression of the transgene in mice

“…Figure 2, 3). Such a large number of transgene copies per genome can be explained by assuming that, along with single integrations of transposons into the genome, insertion of a multicopy concatemer, typical of pronuclear microinjection, could occur (Smirnov and Battulin 2021). Although ddPCR could be imprecise when applied to cases with > 100 copy integrations, we at least could confirm separate integration sites (Sup.…”

“…Unfortunately, in our case, transgenic animals demonstrated only a very low expression level in one of four tested lines. Theoretically, random integration of transgenes could lead to inactivation due to position effect variegation (integration in the inactive locus) or repeat-induced gene silencing (Garrick et al 1998;Smirnov and Battulin 2021). Analysis of multiple founders usually helps to alleviate these problems and find animals with acceptable expression levels.…”

The widely usedUcp1-CreEvdrtransgene elicits complex developmental and metabolic phenotypes

“…For instance, whole genome sequencing and TLA both revealed Adipoq-Cre Evdr transgene inserted into the Tbx18 gene on chromosome 9 19,55,56 , perturbing Tbx18 expression and adding passenger gene copies with possible widespread effects 56 . Additionally, BAC transgenes normally integrate as concatemers leading to multiple full or partial copies of the transgene 3,57,58 . Using ddPCR, we find that Cre coding sequence is present in a single copy in Ucp1-Cre Evdr mice.…”