Mutagenesis in rodents using the L1 retrotransposon

LINE1 (L1) retrotransposons are genetic elements that are present in all mammalian genomes. L1s are active in both humans and mice, and are capable of copying themselves and inserting the copy into a new genomic location. These de novo insertions occasionally result in disease. Endogenous L1 retrotransposons can be modified to increase their activity and mutagenic power in a variety of ways. Here we outline the advantages of using modified L1 retrotransposons for performing random mutagenesis in rodents and discuss several potential applications

Sociability is decreased following deletion of the _trpc4_ gene

Shyness and social anxiety are predominant features of some psychiatric disorders including autism, schizophrenia, anxiety and depression. Understanding the cellular and molecular determinants of sociability may reveal therapeutic approaches to treat individuals with these disorders and improve their quality of life. Previous experiments from our laboratory have identified selective mRNA and protein expression of a nonselective cation channel known as the canonical transient receptor potential channel 4 (TRPC4s) in brain regions implicated in emotional regulation and anxiety. TRPC4 is highly expressed in the corticolimbic regions of the mammalian brain. We hypothesized that robust corticolimbic expression of TRPC4 may regulate the brain’s response to emotion and anxiety resulting in changes in social interaction. Here we test trpc4 gene knockout rats in a model of social anxiety/interaction. We found that the Trpc4 knockout animals spent significantly less time exploring a juvenile intruder rat compared to their wild-type counterparts and Sprague-Dawley (SD) rats. Furthermore, Trpc4 wild-type (Fisher 344) rats explored the juvenile significantly less than the SD rats. These findings indicate that the _trpc4_ gene plays a role in modulating cellular excitability in specific regions of the brain associated sociality and/or anxiety

The SRG Rat, a Sprague-Dawley Rag2/Il2rg Double-Knockout Validated for Human Tumor Oncology Studies

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks

Genome-wide association analysis of genetic generalized epilepsies implicates susceptibility loci at 1q43, 2p16.1, 2q22.3 and 17q21.32

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% and account for 20-30% of all epilepsies. Despite their high heritability of 80%, the genetic factors predisposing to GGEs remain elusive. To identify susceptibility variants shared across common GGE syndromes, we carried out a two-stage genome-wide association study (GWAS) including 3020 patients with GGEs and 3954 controls of European ancestry. To dissect out syndrome-related variants, we also explored two distinct GGE subgroups comprising 1434 patients with genetic absence epilepsies (GAEs) and 1134 patients with juvenile myoclonic epilepsy (JME). Joint Stage-1 and 2 analyses revealed genome-wide significant associations for GGEs at 2p16.1 (rs13026414, Pmeta = 2.5 × 10−9, OR[T] = 0.81) and 17q21.32 (rs72823592, Pmeta = 9.3 × 10−9, OR[A] = 0.77). The search for syndrome-related susceptibility alleles identified significant associations for GAEs at 2q22.3 (rs10496964, Pmeta = 9.1 × 10−9, OR[T] = 0.68) and at 1q43 for JME (rs12059546, Pmeta = 4.1 × 10−8, OR[G] = 1.42). Suggestive evidence for an association with GGEs was found in the region 2q24.3 (rs11890028, Pmeta = 4.0 × 10−6) nearby the SCN1A gene, which is currently the gene with the largest number of known epilepsy-related mutations. The associated regions harbor high-ranking candidate genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. Further replication efforts are necessary to elucidate whether these positional candidate genes contribute to the heritability of the common GGE syndrome

Twin Priming: A Proposed Mechanism for the Creation of Inversions in L1 Retrotransposition

L1 retrotransposons are pervasive in the human genome. Approximately 25% of recent L1 insertions in the genome are inverted and truncated at the 5′ end of the element, but the mechanism of L1 inversion has been a complete mystery. We analyzed recent L1 inversions from the genomic database and discovered several findings that suggested a mechanism for the creation of L1 inversions, which we call twin priming. Twin priming is a consequence of target primed reverse transcription (TPRT), a coupled reverse transcription/integration reaction that L1 elements are thought to use during their retrotransposition. In TPRT, the L1 endonuclease cleaves DNA at its target site to produce a double-strand break with two single-strand overhangs. During twin priming, one of the overhangs anneals to the poly(A) tail of the L1 RNA, and the other overhang anneals internally on the RNA. The overhangs then serve as primers for reverse transcription. The data further indicate that a process identical to microhomology-driven single-strand annealing resolves L1 inversion intermediates

A Novel Active L1 Retrotransposon Subfamily in the Mouse

Unlike human L1 retrotransposons, the 5′ UTR of mouse L1 elements contains tandem repeats of ∼200 bp in length called monomers. Multiple L1 subfamilies exist in the mouse which are distinguished by their monomer sequences. We previously described a young subfamily, called the T(F) subfamily, which contains ∼1800 active elements among its 3000 full-length members. Here we characterize a novel subfamily of mouse L1 elements, G(F), which has unique monomer sequence and unusual patterns of monomer organization. A majority of these G(F) elements also have a unique length polymorphism in ORF1. Polymorphism analysis of G(F) elements in various mouse subspecies and laboratory strains revealed that, like T(F), the G(F) subfamily is young and expanding. About 1500 full-length G(F) elements exist in the diploid mouse genome and, based on the results of a cell culture assay, ∼400 G(F) elements are potentially capable of retrotransposition. We also tested 14 A-type subfamily elements in the assay and estimate that about 900 active A elements may be present in the mouse genome. Thus, it is now known that there are three large active subfamilies of mouse L1s; T(F), A, and G(F), and that in total ∼3000 full-length elements are potentially capable of active retrotransposition. This number is in great excess to the number of L1 elements thought to be active in the human genome

L1 integration in a transgenic mouse model

To study integration of the human LINE-1 retrotransposon (L1) in vivo, we developed a transgenic mouse model of L1 retrotransposition that displays de novo somatic L1 insertions at a high frequency, occasionally several insertions per mouse. We mapped 3′ integration sites of 51 insertions by Thermal Asymmetric Interlaced PCR (TAIL–PCR). Analysis of integration locations revealed a broad genomic distribution with a modest preference for intergenic regions. We characterized the complete structures of 33 de novo retrotransposition events. Our results highlight the large number of highly truncated L1s, as over 52% (27/51) of total integrants were <1/3 the length of a full-length element. New integrants carry all structural characteristics typical of genomic L1s, including a number with inversions, deletions, and 5′-end microhomologies to the target DNA sequence. Notably, at least 13% (7/51) of all insertions contain a short stretch of extra nucleotides at their 5′ end, which we postulate result from template-jumping by the L1-encoded reverse transcriptase. We propose a unified model of L1 integration that explains all of the characteristic features of L1 retrotransposition, such as 5′ truncations, inversions, extra nucleotide additions, and 5′ boundary and inversion point microhomologies

Progressive Saturation Improves the Encapsulation of Functional Proteins in Nanoscale Polymer Vesicles

Purpose To develop a technique that maximizes the encapsulation of functional proteins within neutrally charged, fully PEGylated and nanoscale polymer vesicles (i.e., polymersomes). Methods Three conventional vesicle formation methods were utilized for encapsulation of myoglobin (Mb) in polymersomes of varying size, PEG length, and membrane thickness. Mb concentrations were monitored by UV–Vis spectroscopy, inductively coupled plasma optical emission spectroscopy (ICP-OES) and by the bicinchoninic acid (BCA) assay. Suspensions were subject to protease treatment to differentiate the amounts of surface-associated vs. encapsulated Mb. Polymersome sizes and morphologies were monitored by dynamic light scattering (DLS) and by cryogenic transmission electron microscopy (cryo-TEM), respectively. Binding and release of oxygen were measured using a Hemeox analyzer. Results Using the established “thin-film rehydration” and “direct hydration” methods, Mb was found to be largely surface-associated with negligible aqueous encapsulation within polymersome suspensions. Through iterative optimization, a novel “progressive saturation” technique was developed that greatly increased the final concentrations of Mb (from  2.0 mg/mL in solution), the final weight ratio of Mb-to-polymer that could be reproducibly obtained (from  4 w/w% Mb/polymer), as well as the overall efficiency of Mb encapsulation (from  90%). Stable vesicle morphologies were verified by cryo-TEM; the suspensions also displayed no signs of aggregate formation for > 2 weeks as assessed by DLS. “Progressive saturation” was further utilized for the encapsulation of a variety of other proteins, ranging in size from 17 to 450 kDa. Conclusions Compared to established vesicle formation methods, “progressive saturation” increases the quantities of functional proteins that may be encapsulated in nanoscale polymersomes.National Institutes of Health (U.S.) (1R43CA159527-01A1 and 1R43AI096605-01)Kentucky Science and Technology Corporation (KSTC-18-OCIS-194, KSTC-184-512-12-135, KSTC-184-512-13-156)Charles W. and Jennifer C. Johnson Koch Institute Clinical Investigator AwardKathryn Fox Samway FoundationMisrock Foundatio