Lentiviral Mediated Transgenesis by In Vivo Manipulation of Spermatogonial Stem Cells

This report describes a technique for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells with a high rate of success. Spermatogonial stem cells (SSCs) in pre-pubescent animals were infected in vivo with recombinant lentiviruses expressing EGFP-f and mated with normal females. All male pre-founder mice produced transgenic pups with an overall success rate of over 60%. The transgene was heritable and the pre-founder mice could be used in multiple mating experiments. This technology could be used to perform overexpression/knockdown screens in vivo using bar-coded lentiviruses, thus permitting the design of genetic screens in the mouse. Further, this technology could be adapted to other laboratory animals resulting in the generation of model systems that closely approximate human development and disease

Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis

Percentage of EGFP-f positive pups obtained from individual matings with three different pre-founder mice.

<p>The pre-founder mice were mated with multiple WT female mice and the frequency of EGFP-f positive pups determined after each mating. Note that an overall success rate of greater than 60% was obtained in these matings.</p

List of oligonucleotides used for PCR reactions.

<p>List of oligonucleotides used for PCR reactions.</p

Analysis of integration events in the EGFP-f transgenic mice.

<p><b>A</b>. Protocol for the identification of integration sites. <b>B</b>. Graph showing the frequency of integration events observed in the transgenic animals. Chromosome number is on the X-axis and the number of mice showing integration in the respective chromosomes is on the Y-axis. The number above each bar in the graph indicates the percentage of total integration events analyzed on each chromosome. <b>C</b>. Graph showing the number of integration events in each mouse. No of integration events is on the X-axis and number of mice on the Y-axis. The number above each bar indicates the percentage of mice with one, two or three integration events.</p

Summary of the integration events observed in the different transgenic animals.

<p>The chromosome number is shown in column one while the number of the individual mice with an integration in these chromosomes is shown in column 4. The integration events in each chromosome are shown in the third column of this table with the appropriate accession number in column 2. As can be seen from the table, multiple mice show different combinations of integration events suggesting that transgene expression is independent of integration site. The number of assigned to the mice in this table is similar to the number reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021975#pone-0021975-g001" target="_blank">Figure 1</a>.</p

K8 downregulation leads to an inhibition of transformation in vitro and in vivo.

<p><b>A.</b> The S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. <b>B.</b> 10⁶ cells from the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were injected subcutaneously into 5 different nude mice and tumor size determined every week as described. Tumor volume is plotted on the Y-axis and the time in weeks on the X-axis. <b>C.</b> Protein extracts from primary tumors from mice injected with the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 and β-actin. The numbers indicate different mice injected with the single or double knockdown clones. All the samples were run on the same gel and the Western blots performed at the same time. <b>D.</b> Haematoxylin and eosin staining of paraffin embedded sections of lung tissue from nude mice injected with 10⁶ cells of the vector alone (S10P3) or the double knockdown clones (8.21 and 8.28). Lung section from mice injected with S10P3 cells show extensive metastasis with thickening of alveolar walls from deposition and aggregation of metastasized tumor cells, whereas lungs from mice injected with the double knockdown cells show normal lungs with thin walled alveoli, with a few metastatic tumor cells. The images in the top row are at magnification x100 and images in the bottom row are at magnification x 400. <b>E.</b> PCR reactions were performed on DNA isolated from paraffin sections for the presence of Alu repeats in genomic DNA. Genomic DNA was purified from normal lung tissue, lung tissue from mice injected with cells with PKP3 knockdown alone (S10P3) and lung tissue from mice injected with the double knockdown clones (8.21 and 8.28). Lung tissues from uninjected mice (N) were used as a negative control for the Alu PCR. A PCR for the mouse patch gene was performed as a loading control. <b>F.</b> Immunohistochemical staining was performed with antibody against K8 on sections of paraffin embedded tissue of tumor and lungs of mice injected with S10P3 vector control cells or the double knockdown clones, 8.21 and 8.28. Images a, b, c, g, h and i are taken at a magnification x 200 and the respective magnified images at magnification x 400, of the indicated areas in the white rectangles, are represented by images d, e, f, j, k and l. Tumor tissue from mice injected with S10P3 (a and d) show K8 staining at levels higher compared to tumors from mice injected with the double knockdown clones, 8.21 and 8.28 (b, c, e and f). In contrast lung tissue, from mice injected with the vector control S10P3 cells (g and j) and the double knockdown clones (8.21 and 8.28) (h, i, k and l) show elevated K8 staining in metastatic areas of the lungs with infiltrated tumor cells.</p

High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.

<p><b>A.</b> Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. <b>B.</b> S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to K8 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038561#pone.0038561-Gosavi1" target="_blank">[28]</a>. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. <b>C.</b> A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. <b>D.</b> Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. <b>E.</b> Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. <b>F.</b> HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.</p