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

Deletion of the trpc4 gene and its role in simple and complex strategic learning

The TRPC4 ion channel is expressed extensively in corticolimbic and a subpopulation of midbrain dopamine neurons. While TRPC4 knockout (KO) rats exhibit reduced sociability and social exploration, little is known about the role of TRPC4 in motivation and learning. To identify a function for TRPC4 channels in learning processes  we tested TRPC4 KO and normal wild type (WT) rats. TRPC4 KO and WT rats exhibited no differences in Y-­maze learning or simple discrimination learning. Furthermore, on a more complex serial reversal shift task designed  to assess strategic learning where the reward and non-­reward cues were repeatedly reversed between training sessions both TRPC4 KO and WT rats   performed equally well. Finally, we found no   performance differences when using a conditional reversal shift task where a tone signals the reversal of reward and non-reward cues within sessions. These data suggest that although TRPC4 channels may play a role in social interaction/anxiety  they exert a minimal role in simple and complex strategic learning

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

Insights into the mechanism of *L1 retrotransposition

L1 retrotransposons have had a tremendous impact on mammalian genomes through a variety of mechanisms including retrotransposition, transduction of flanking sequence, and mobilization of non-autonomous retrotransposons in trans. The significance of L1 elements is becoming increasingly apparent, but the mechanism of L1 retrotransposition remains poorly understood. In this thesis, I describe the creation of an Enhanced Green Fluorescent Protein (EGFP) based retrotransposition cassette and demonstrate that is can be used to detect near real-time retrotransposition in a single cell. I use the cassette to tag human L1 elements and test the elements in a new cultured cell assay. I next describe the creation of a mouse model of human L1 retrotransposition using tagged human L1 elements. Retrotransposition in the male germ line of transgenic mice occurs at frequency as high as 1 in 100 sperm. The L1 promoter is germ line-specific and retrotransposition often occurs in late meiosis and post-meiosis. The mouse model is valuable as a system to study L1 retrotransposition in vivo and may pave the way to an eventual random mutagenesis system in mouse. In a subsequent chapter, I describe a bioinformatics project that has provided insight into several phenomena of L1 retrotransposition. First, I show that L1-mediated transduction is a common process and that it may have created up to 0.6% of the mass of the genome. L1-mediated transduction may be a source of genetic diversity as it may move exons or regulatory regions to new areas of the genome. Second, I demonstrate that L1 elements in the genome frequently show inversion and truncation of their 5′ ends. I hypothesize that L1 inversion is a consequence of its mechanism of insertion, a coupled process of reverse transcription and integration called target primed reverse transcription. I propose a model called “twin priming” to explain the inversions and provide evidence in support of the model. In the last chapter, I describe the discovery of an active SVA retrotransposon. I demonstrate that SVA elements are active non-autonomous human retrotransposons and that they are mobilized by using the L1 retrotransposition machinery in trans