Production of Transgenic Pigs Mediated by Pseudotyped Lentivirus and Sperm

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency

Anodic, Cathodic, and Annihilation Electrochemiluminescence Emissions from Hydrophilic Conjugated Polymer Dots in Aqueous Medium

Hydrophilic poly­[2-methoxy-5-(2-ethyl­hexyloxy)-1,4-phenylene­vinylene] (MEH-PPV) conjugated polymer dots (CP-dots) capped by Triton X-100 were synthesized. For the first time, the electrochemiluminescence (ECL) emission of CP-dots was investigated in aqueous solution. At the glassy carbon/water interface, the CP-dots have excellent and multichannel ECL properties, such as having annihilation ECL activity in the absence of coreactants, and give bright anodic and cathodic ECL emission (590 nm) in the presence of tri-<i>n</i>-propylamine (TPrA) and peroxydisulfate (S₂O₈^2–), respectively. The versatile ECL properties of the hydrophilic CP-dots combined with their low cytotoxicity, good biocompatibility, and easy bioconjugation may suggest promising applications of this new type of ECL nanomaterial in novel biosensing and bioimaging, and new types of light-emitting devices

Map of lentiviral vector.

<p>Map of lentiviral vector.</p

Absorption of lentiviral particles by pig spermatozoa under a scanning electronic microscope and immunochemical detection of lentiviral particles on spermatozoa.

<p>A: lentiviral particles (40000×). B: Two lentiviral particles are clearly attached on the head of one spermatozoa (10000×). C: Two lentiviral particles absorbed on a spermatozoa tail (25000×). D: Observation of interaction between a lentiviral particle and the spermatozoa surface in an enlarged image (60000×). E: A lentiviral particle entering a spermatozoon (7500×). Immunuchemical detection of lentiviral particles attached on sperm was performed using a monoclonal mouse anti-VSV-Glycoprotein antibody, F was view field of porcine spermatozoa (200×), and red fluorescence was clearly observed emitted at 570 nm (G, 200×).</p

PCR amplification of transgene DNA sequences in piglets.

<p>PCR amplification of transgene DNA sequences in piglets.</p

Number of lentiviral particles associated with sperm cells after incubation (n = 3).

<p>Number of lentiviral particles associated with sperm cells after incubation (n = 3).</p

RT-PCR amplification of a specific fragment of the lentiviral vector after incubation with sperm cells.

<p>Lane M: DNA Ladder DL2000 (2000, 1000, 750, 500, 250 and 100 bp from top to bottom). Lane 1: Negative control with H₂O as template. Lane 2: Negative control with total RNA extracted from sperm alone as template, both of which showed no PCR products. Lanes 3–6: Specific PCR products were detected in sperm samples incubated with lentiviral particles for 2, 4, 6 and 8 h, respectively.</p

Identification of transgenic pigs by Southern blot analysis.

<p>Southern blot was performed under optimized condition with a Dig-lableled probe of 649 bp fragment. Samples in lanes 1 and 2 are from PCR-positive the founder No. 18 and No. 41, respectively, lanes 3–8 are their offsprings; The specific hybridization signal was detected in Lanes 1, 2, 4, 9; lanes 3, 5, 6, 7, 8 are negative, respectively; lane 10 is wild type control; lane 11 is positive controls (The plasmid pLV-siRNA); M is DNA Molecular Weight VII (DIG-labeled Roche).</p