Characterisation of Genes Identified During a RADES-PCR Screen of Concanavalin A-Treated Procyclic Trypanosoma brucei rhodesiense

Previous studies demonstrated that procyclic Trypanosoma brucei could be induced to die by culturing cells in the presence of the lectin Concanavilin A, and that throughout the three day death process de novo gene expression could be detected. The current study utilised a differential display reverse transcription polymerase chain reaction (DDRT-PCR) technique known as randomly amplified differentially expressed sequences (RADES) PCR to identify cDNAs whose expression levels fluctuated during the time-course of death. 64 PCR products were detected, of which 27 were reamplified and sequenced. Database searches were conducted using the BLAST algorithm, identifying 17 significant matches with known genes in the database. 8 of these encoded novel T. brucei genes. Northern blot analysis was attempted in order to confirm expression patterns indicated by RADES-PCR. However, the data obtained was inconclusive due to lack of a marker constitutively expressed during cell death. Two genes identified during this study, QM and MOB1, were characterised further. The human QM gene was first identified as a transcript upregulated in a non- tumourigenic Wilms' tumour microcell hybrid relative to the parental cell line, and subsequent experiments suggested that the QM gene encoded a transcription factor. More recent evidence indicates that QM is actually a ribosomal protein associated peripherally with the 60S ribosomal subunit. During the current study Southern blot analysis was conducted, indicating the presence of 2 copies of the T. brucei QM gene, one of which was isolated from a genomic lambda library. Sequence analysis revealed 60% amino acid identity between the T. brucei QM and QM homologues from diverse eukaryotes. A recombinant epitope-tagged QM was inducibly expressed in procyclic T. brucei. Indirect immunofluorescence microscopy revealed nuclear exclusion and colocalisation with GPI8, a component of the transamidase complex responsible for glycosylphosphatidylinositol (GPI) anchor attachment which is hypothesised to localise to the endoplasmic reticulum. Tagged QM in cellular extracts was demonstrated to be insoluble following lysis in a 1% Triton X-100 buffer, suggesting an association with a large protein complex. Taken together these results suggest that the T. brucei QM is a ribosomal protein. MOB1 is an essential yeast gene required for completion of mitosis and maintenance of ploidy. While a number of interacting partners have been identified for MOB1, the function of this protein remains unknown. During the current study Southern blot analysis showed the presence of 2 copies of the T. brucei MOB1 gene within 12 kb of each other. Both of these genes were isolated from a single genomic X clone and were named MOB1-1 and MOB1-2. Sequence analysis revealed that while >96% of the amino acid sequence encoded by MOB1-1 was conserved in the MOB1-2 gene, the latter had a predicted N-terminal extension of 82 residues. Inducible expression of an antisense MOB1-1 mRNA in procyclic T. brucei resulted in a significant reduction in proliferation, indicating a role for MOB1 in cell cycle progression. A recombinant epitope-tagged MOB1-1 was inducibly expressed in procyclic T. brucei. Indirect immunofluorescence microscopic analysis revealed a homogenous distribution throughout the cell, allowing no insight into function. A recombinant MOB1-1 polypeptide was produced in E. coli and used to inoculate rabbits. Resultant antiserum detected a T. brucei protein of the predicted size of MOB1-1. This antiserum will prove invaluable for future studies, allowing the subcellular location of native MOB1-1 to be established, and purification of interacting partners through co-immunoprecipitation to be carried out

Case reports: pig 917.

<p>Grouped data for the transgenic pig 917 (3 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 917 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–P) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), GFAP (H,I) and S-opsin (I,J,O,P) in the central region of the retina of pig 917 (E–J) and a non-transgenic control (K–P). Arrows in E, G, K and M show examples of M-opsin positive outersegment, arrowhead in G shows displaced nuclei, arrowhead in J shows a displaced nucleus in a S-opsin positive cell. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–J represents 50 µm.</p

Case reports: pig 920.

<p>Grouped data collected for the transgenic pig 920 (4 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 920 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–L) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), and GFAP (H,I) in the central region of the retina of pig 920 (E–H) and a non-transgenic control (I–L). Arrows in E, G, I and K show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–L represents 50 µm.</p

Expression of GUCY2D^E837D/R838S transcript in transgenic pigs.

<p>Assessment of transgene expression by RT-PCR. Transgenic pigs: 904, 907, 908, 914, 915, 917, 918, 920, control non-transgenic pig (929). +: with reverse transcription; −: without reverse transcription; hGUCY2Dmut: GUCY2D^E837D/R838S PCR fragment; pig GUCY2D: pig GUCY2D PCR fragment; GAPDH: pig GAPDH PCR fragment.</p

Range of visual function in GUCY2D^E837D/R838S transgenic pigs.

<p>(A) Traces of photopic electroretinogram recordings (ERG) at 11 weeks are shown for one representative control and several transgenic animals. (A, first column) single flash at 3 cds/m², (A, second column) single flash at 10 cds/m2, (A, third column) flickers at 3 cds/m²,30 Hz. (B) b-wave amplitudes for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (C) a-wave amplitudes obtained for all examined animals at 11 weeks with single flash at 3 cds/m² and at 10 cds/m². (D) Representation of the time needed to complete the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. (E) Representation of the errors (missing or striking into an obstacle), alternative prospections (the number of times individuals investigated an obstacle by sniffing or licking) and resulting scores from the obstacle course at 24 and 52 weeks for transgenic and non-transgenic control animals. Horizontal bars in B,C represent the mean of the different groups with the SEM; *: p<0.05;***: p<0.001; errors in E: miss or strike into an obstacles; alternative prospection in E: sniff or lick the obstacles; score in E: sum of errors and alternative prospections.</p

Abnormal retinal morphology in transgenic pigs.

<p>(A) Morphological examination of a retina from a control animal reveals retinal layers: outer segments (OS), inner segment of photoreceptors (IS), photoreceptor nuclei (ONL). (B) Displaced nuclei were observed in the outer segment layer in transgenic retina (arrows). (C) Quantification of the number of displaced nuclei in transgenic and control animals. (D,E,F) Immunolabeling for specific cone markers PNA and M-opsin in transgenic retina identified most of these displaced cells as cones. (G,H) Immunolabeling for specific cone markers PNA and M-opsin in control retina. (I) Quantitation of relative density of displaced cones as determined by PNA or M-opsin labeling across 100 µm on the section. OS: outer segment; IS: inner segment; ONL: outernuclear layer; PNA: peanut agglutinin; M-opsin: medium wavelength opsin; nb: number. Scale bar in A, B and D to F represents 50 µm.</p

Case report: pig 908.

<p>Grouped data for the transgenic pig 908 (2 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 908 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). For technical reasons, the time to reach the end of the obstacle course has not been measured at 52 weeks. (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–G) Immunolabeling for M-opsin (E), PNA (F) and merged picture (G) in the central region of the retina of pig 908. Arrows in E and G show examples of M-opsin positive outersegment, arrowhead in G shows a displaced nucleus. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–G represents 50 µm.</p

Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients