Yin Zhi, Guo Jia, Bou Gerelchimeg, Liu Shi-chao, Mu Yan-shuang, and Liu Zhong-hua

College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

Lentivirus Mediated Gene Manipulation in Trophectoderm of Porcine Embryos

Yin Zhi, Guo Jia, Bou Gerelchimeg, Liu Shi-chao, Mu Yan-shuang, and Liu Zhong-hua*

College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm (TE) lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA (shRNA) and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.

pig, trophectoderm, lentivirus, gene manipulation

Introduction

Pig is important farm animal and potentially useful in human disease model. However, deficiency of fundamental research in porcine embryonic development and related mechanism severely lagged pace of the adoption of pig as a model in human disease research. Even the essential regulating gene functions are clear during the formation of trophectoderm and inner cell mass in mouse blastocyst, unfortunately, the related knowledge and research in other large animal models is lacking. Up to data, preliminary results indicated that expression patterns such as CDX2 and OCT4 in embryos of livestock including pig, cattle and goat were different from the pattern in mouse embryo. Conventional transgenic approaches such as somatic cell nuclear transfer and pronuclear injection manipulate the genome of whole embryo, so that it is hard to define the roles of genes in a specific cell lineage of embryo. As we all known, porcine blastocysts sustain and continuously develop for at least 3 days in vitro, since the cavitation. Thus, porcine embryo during this period is a ready model in the study of trophectoderm (TE) formation and development compared to mouse embryos, which only sustain for 1-2 days in vitro before implantation (Kuijk et al., 2008). Porcine trophoblastic could provide a powerful model for understanding trophoblast cell biology as well as placental gene expression and proteomics in vitro. For this reason, we developed a lentivirus mediated TE specific gene manipulating method in porcine embryo. This method would facilitate gene expression control and the relatedstudies on TE developmental process.

Materials and Methods

Unless otherwise stated, all the chemicals were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA).

Preparation of lentivirus vectors

The constitutive and inducible lentivirus vectors were constructed based on commercial vectors (Addgene) FUW-M2rtTA and FUW-tetO-hOCT4. In order to construct FUW-EGFP, FUW-Cdx2DsRed, FUW-miR30-Oct4shRNA-EGFP, using primers flagged by Xba I and Eco RI, EGFP, Cdx2DsRed and EGFP-miR30-Oct4shRNA were cloned from plasmid pEGFP-C1 and our formerly constructed plasmids pCMV-CDX2DsRed and pGW-EGFP-miR30-Oct4shRNA and replaced the original open reading frame-rtTA behind hUbC promoter in FUW-M2rtTA. In order to construct FUW-tetO-EGFP, FUW-tetOCdx2DsRed, and FUW-tetO-miR30-Oct4shRNAEGFP, those fragments were cloned and used to replace the original open reading frame-hOCT4 behind TRE element in FUW-tetO-hOCT4.

Lentivirus production

In order to obtain high titter virus, those lentivirus plasmids and packing plasmids were transfected into 293T cells using liposome method. After 48 h transfection, virus-containing supernatant was harvested, centrifuged at a low speed (2 000 r ? min-1for 10 min), and filter purified with a Millipore Stericup filter unit (Millipore, Billerica, MA). Concentrated virus particles were then aliquoted and stored at–80℃.

IVF procedure

Oocytes were obtained and matured in vitro as described previously (Liu et al., 2008). The matured COCs was vortexed for 3 min in Hepes-buffered medium with 0.1% hyaluronidase to remove the cumulus cells. Denuded oocytes were washed and held in the modified Tris-buffered medium (mTBM) prior to fertilization (Abeydeera et al., 1997). Each 30 oocytes were delivered to 50 μL of mTBM drop under oil and held in 5% CO2in air at 39℃. The semen was washed three times in DPBS with 0.1% BSA and after centrifugation for 4 min at 1 900 g the sperm pellet was resuspended in 1 mL mTBM. Following concentration measurement with hemocytometer, resuspension was adjusted to the optimal concentration with additional mTBM. 50 μL of the final sperm dilution was added to the oocytes and incubated for 5 h in 5% CO2in air at 39℃. The presumptive zygotes were then washed three times and incubated in PZM3 embryo culture medium (Yoshioka et al., 2002) in 5% CO2in the air at 39℃.

TE specific lentivirus transduction

On the 5th day of the culture, the cavitated early blastocysts were treated with pronase for 10 min, so that the zona was removed and used to incubate with lentivirus. Forlentivirus mediated TE specific infection, the virus were diluted with CO2equilibrated PZM3 to 1×107titer. 2 μL of diluted virus preparation was added to 8-μL droplets under oil containing up to 10 expanded blastocysts. The embryos were cultured with virus for 3 h, then washed by serially transferring using a pipette into fresh PZM3 droplets of at least five times and finally transferred into 500 μL PZM3 medium (for the inducible system, and 2 μg ? L-1DOX was added in the culture medium).

lmmunofluoresence

Embryos were fixed in 4% PFA for 420 min at room temperature and treated for routine immunofluorescence. Briefly, to visualise OCT4, fixed embryos were permeabilised in 0.1% Triton X-100 in PBS for overnight at 4℃ and blocked in 2% BSA in PBS for 1 h. Goat anti OCT4 (SantaCruz) at 1 : 100 and secondary AlexaFluor 488-conjugated anti-goat antibody or secondary AlexaFluor 546-conjugated antigoat antibody at 1 : 1 000 were used. After antibody incubations and washes, embryos were stained withHoechst33342, and mounted on slides. Cells were imaged on a Leica TS2 confocal microscope.

Results

Construction of lentivirus vectors

In order to prove the general usage of our procedure in lentivirus mediated TE specific gene manipulation, we constructed six lentivirus vectors, including constitutive expressing vectors: FUW-EGFP, FUWCdx2DsRed, FUW-miR30-Oct4shRNA-EGFP and their corresponding tetracycline inducible vectors: FUW-tetO-EGFP, FUW-tetO-Cdx2DsRed, FUW-tetO-miR30-Oct4shRNA-EGFP. The construction of vectors was conducted as the describtion in materials and method sections. The vector backbones were obtained via restriction endonuclease digestion with Xba I and Eco RI and other expressing fragments and elements were obtained by PCR method (Fig. 1). The vector maps of the lentiviral construction are shown in Fig. 2.

TE specific expression of fluorescent marker in porcine blastocyst after lentivirus transduction

The procedure for transduction of lentivirus for constitutive and inducible vectors was conducted as the workflow shown in Fig.3. At 12 h transduction or induction, the green fluorescence of EGFP protein from FUW-EGFP and FUW-tetO-EGFP lentivirus could be observed clearly under 488 nm light (Fig. 4a). Further confocal microscopy observation proved that lentivirus specifically infected the outer layer-TE cells, but the inner sites-ICM (Fig. 4b).

Fig. 1 Obtaining of vector backbone via restriction endonuclease digestion and PCR cloning of expressing fragments used for this study

Functional genes were upregulated or downregulated exactly in TE

Then, in order to test the efficiency of our system in functional gene manipulation, we infected 5D blastocyst with lentivirus FUW-Cdx2DsRed, FUW-miR30-Oct4shRNA-EGFP and FUW-tetO-Cdx2DsRed, FUW-tetO-miR30-Oct4shRNA-EGFP. CDX2 was chosen as a candidate gene to be overexpressed in TE. CDX2 is a major transcription factor controlling TE development (Jedrusik et al., 2008; Wu et al., 2010; Strumpf et al., 2005), its protein is localizing in cell nucleus of TE following CDX2 overexpressing lentivirus infection. As CDX2 and fluorescent protein DsRed were expressed as a fusion protein, the red fluorescence is the indicator of letivirus derived CDX2 localization. As shown in Fig. 5a, CDX2DsRed was expressed in nucleus of TE. Except TE specific gene overexpression, we also constructed OCT4 targeted shRNA expressing vectors FUW-miR30-Oct4shRNA-EGFP and FUW-tetO-miR30-Oct4sh-RNA-EGFP for confirming the performance of our system in gene interference. The immunofluorescence result showed that OCT4 expression in TE was significantly downregulated, when its expression in ICM was not influenced (Fig. 5b). OCT4 is a well known pluripotent gene which is exclusively expressed in ICM of mouse blastocyst (Loh et al., 2006; Niwa et al., 2005). However, OCT4 is also expressed in TE of the porcine blastocyst for long period (Spencer et al., 2006; Vejlsted et al., 2006; Kirchhof et al., 2000). The reason for OCT4 expression in porcine TE is still unclear. Our system might provide a possibility to investigate OCT4 function in porcine TE.

Fig. 2 Construction of constitutive and inducible lentiviral vectors

Fig. 3 Scheme illustration of porcine TE specific lentivirus infection

Fig. 4 12 h post FUW-EGFP and FUW-tetO-EGFP lentivirus infection (DOX induction), GFP specifically expressed in TE

Fig. 5 TE specific functional gene manipulation

Discussion

Lentivirus based method has been used to investigate gene functions in embryonic development and first lineage segregation in mouse. In 2007, three independent groups reported the promising technology in mouse placenta specific gene manipulation by transducing zona-free blastocyst using lentiviral vector (Okada et al., 2007; Malashicheva et al., 2007). With this method, transgenic mouse placentas can be generated at 100% of efficiency while all the fetuses remain non-transgenic. It was considered as a robust system for studying the placental organogenesis with implications for the treatment of placental dysfunction and tranducing lentiviral vectors expressing short hairpin shRNAs for trophoblast-specific knockouts. Application of this method substantially rescued mice deficient in Ets2, Mapk14 and Mapk1 from embryonic lethality caused by placental defects (Okada et al., 2007). Soon after, new techniques like integrasedefective lentiviral vectors with Cre/LoxPsystem (Morioka et al., 2009) and bioluminescence imaging system have been integrated for special application (Fan et al., 2011). Then, in 2011, researchers also creatively obtained induced pluripotent stem cells from trophoblast cells in blastocysts which were incubated with four classic factors (Oct4, Sox2, c-Myc and Klf4) bearing lentivirus (Kuckenberg et al., 2011). In 2013, Zhou et al. using this technique proved the essential role of the proprotein convertase furin in trophoblastsy ncytialization. The pig has been considered for a long time among the best models for medical, clinic and fundamental biology. However, the lack in authentic embryonic stem cells greatly impacted the scientific contribution of this species. One of the major reasons lagged the progress of porcine ESC establishment is the limited knowledge on porcine embryonic development, lineage segregation and the related cell signaling pathways. It is believed that enrichment in methodology of porcine developmental biology is crucial. Our method for TE specific gene manipulation in porcine embryo would also facilitate the further studies surrounding mechanisms about inner cell mass (ICM) and TE derivation, maintenance and interaction. Knowledge in these fields could offer implication for porcine ESC establishment.

Conclusions

In the present study, we developed a lentivirus based method to manipulate gene expression in porcine TE. The present study provided valuable information for the further functional verification of genes in porcine TE, and proposed a new approach to further investigate the mechanism of porcine embryo development.

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S828; Q78

A

1006-8104(2014)-03-0039-07

Received 7 March 2014

Supported by the Scientific Research Fund of Heilongjiang Provincial Education Department (11551039)

Yin Zhi (1981-), male, Ph. D, engaged in the research of transgenic animals. E-mail: yinzhineau@126.com

* Corresponding author. Liu Zhong-hua, professor, supervisor of Ph. D student, engaged in the research of animal embryo engineering. E-mail: liu086@126.com