lentivirus mediated gene modification with placenta specificity

I am quite sure this technique exists. Today, I finally find a paper describing it. However, the full text is in Japanese, which I cannot fully understand. From the numbers in the text, I guess it does not have a method section. :-(

Yakugaku Zasshi. 2008 Jul;128(7):1007-11.
Development and Evaluation of a Novel Placenta-specific Gene Manipulation Method Using Lentiviral Vectors.
Okada Y.

Research Institute for Microbial Diseases, Osaka University.

The placenta plays numerous important roles to support fetal development such as gas exchange, nutrient supply, and hormone production. Placental defects underlie many aspects of pregnancy losses and complications; thus understanding and regulating gene function during placentation is of high clinical relevance. However, the lack of a facile and efficient method for placenta-specific gene manipulation has hampered study of the placenta. We have previously shown that transduction of fertilized mouse eggs with lentiviral (LV) vectors efficiently generates transgenic animals; however, transgene expression occurred in both the fetus and the placenta. In the present study, we transduced zona-free blastocysts with LV vectors expecting placenta-specific gene expression, since most placental cells differentiate from trophoblast cells that form the outermost layer of the blastocyst. Transgene expression was observed in trophoblast cells from preimplantation stages and in the placenta throughout gestation. All the analyzed placentas carried the transgene, while none of the fetuses became transgenic. By applying this method, embryonic lethality caused by placental defects in several knockout animal models was substantially rescued. This technology provides a powerful system for gene manipulation exclusively in placental organogenesis with implications for the treatment of placental dysfunction.

PMID: 18591868

This one figure should summary the central idea of this paper. Clean and Beautiful.

Update, after some reading/googling, I realize that there was a similar paper from him published last year in Nature Biotechnology already! I might miss it before.

Nat Biotechnol. 2007 Feb;25(2):233-7.
Complementation of placental defects and embryonic lethality by trophoblast-specific lentiviral gene transfer.
Okada Y, Ueshin Y, Isotani A, Saito-Fujita T, Nakashima H, Kimura K, Mizoguchi A, Oh-Hora M, Mori Y, Ogata M, Oshima RG, Okabe M, Ikawa M.

Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.

Placental dysfunction underlies many complications during pregnancy, and better understanding of gene function during placentation could have considerable clinical relevance. However, the lack of a facile method for placenta-specific gene manipulation has hampered investigation of placental organogenesis and the treatment of placental dysfunction. We showed previously that transduction of fertilized mouse eggs with lentiviral vectors leads to transgene expression in both the fetus and the placenta. Here we report placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts after removal of the zona pellucida. All of the placentas analyzed, but none of the fetuses, were transgenic. Application of this method substantially rescued mice deficient in Ets2, Mapk14 (also known as p38alpha) and Mapk1 (also known as Erk2) from embryonic lethality caused by placental defects. Ectopic expression of Mapk11 also complemented Mapk14 deficiency during placentation.

PMID: 17220877

The parts I am most interested include:

• Lentiviral vector production. HIV-I based, self-inactivating, lentiviral vector plasmid pLV-EGFP was constructed by replacing the PGK promoter with the CAG promoter fragment in pRRLsin-hPGK-EGFP2, 7. Other constructs such as pLV-Ets2, pLV-Mapk14, pLV-Mapk11 and pLV-Mapk1 were generated by replacing the EGFP fragment of pLV-EGFP with PCR-amplified murine cDNAs. VSV-G pseudotyped lentiviral vectors were generated as described7. Briefly, 293T cells were transfected with the pLV plasmid, packaging plasmid and VSV-G- expressing plasmid by the calcium phosphate method. Lentiviral vectors were harvested at 2 and 3 days after transfection, and then concentrated 1,000 times by ultracentrifugation (50,000g, 2 h times 2 times). After resuspension with HBSS buffer, lentiviral vector concentration was determined by measuring p24 gag antigen by ELISA (Retrotek, Zeptometrix).
• Transduction of preimplantation embryo. Wild-type or heterozygous mutant female mice with B6D2F1 background were superovulated by intraperitoneal injection of pregnant mare’s serum gonadotropin (5 units) followed by human chorionic gonadotropin (5 units) 48 h later and then mated with wild-type or heterozygous mutant males respectively. Two cell–stage embryos were collected from the females at 1.5 days after copulation, and then incubated for 2 days to obtain blastocysts. Prior to lentiviral vector transduction, the zona pellucida was removed by treatment with acidic Tyrode’s solution8. Zona pellucida–free embryos were incubated individually in 5 mul of medium containing lentiviral vector (1 times 103 ng p24/ml) for 2 days for two-cells or 4 h for blastocysts. For blastocoel injection, lentiviral vector (1 times 104 ng p24/ml) was injected into blastocoel using an Eppendorf Transjector 5246 micromanipulator11. Transduced blastocysts were observed under a fluorescence microscope (KEYENCE, BZ-8000) or implanted into pseudopregnant females. Blastocyst images were obtained by computer-assisted Z-stack and haze reduction.
• Tetraploid complementation. Tetraploid embryo and aggregation chimera were prepared as described previously8. In brief, B6D2F1 two cell–stage embryos were placed in fusion buffer and electrofusion was performed by applying 140 V for 50 mus after aligning embryos between the electrodes. A wild-type tetraploid four-cell embryo and a mutant diploid eight-cell embryo were aggregated and then the developed blastocysts were implanted into pseudopregnant females.
• Fluorescence in situ hybridization analysis (FISH). pLV-EGFP plasmid DNA was labeled with digoxigenin-11-dUTP by nick translation for use as a probe. Slides were denatured for 2 min in 70% formamide/2 times SSC at 70 °C and dehydrated. The probe was denatured for 10 min in at 75 °C and applied to the slides. After hybridization, the slides were washed in 50% formamide/2 times SSC at 37 °C and 1 times SSC at 20 °C for 20 min each. Detection of the probe signals was performed with Cy3-labeled anti-digoxigenin. The FISH images were captured under a Leica DMRA2 microscope with the CW4000 FISH application program of Leica Microsystems Imaging Solution Ltd.