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

<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.</span>

669-676The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), <span style="mso-bidi-font-weight: bold">using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo. </span

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

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

List of oligonucleotides used for PCR reactions.

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