Performing Department
Animal Dairy & Veterinary Scie
Non Technical Summary
Cloning is an important tool in many research fields and holds great promise for the creation of domestic animals with high genetic value. However, the animal production efficiency of cloning remains low. Egg quality plays a critical role in the efficiency of embryo and fetus development. A significant determinant of egg quality is the ability to polyadenylate genetic message to allow egg development. Polyadenylation is the process of adding a protective and transport-friendly tail on the end of the genetic message, stabilizing it so that can be efficiently read by the cell's protein-making machinery. Our working hypothesis is that low quality eggs inefficiently polyadenylate genetic messages and that increasing egg polyadenylation will improve egg quality and the efficiency of cloning. We will test this hypothesis by adding extra protein, which controls the polyadenylation process (CPEB1), in cattle cloned eggs and evaluate the effects on embryo quality and gene expression in cloned embryos. With discoveries made in this work on the role of polyadenylation during cloned embryo development, we have great potential to create a new approach to improve oocyte quality and development of cloned embryos,leading to improvements in cattle cloning that will increase production efficiency.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to improve cloning efficiency by overexpressing CPEB1 in bovine cloned embryos. The overall objectives of this proposal are to determine the role of CPEB1 in bovine embryogenesis and identify its effects on gene expression profiles in cloned embryos injected with CPEB1.The three objectives are:1. Determine CPEB1 expression levels at different developmental stages of IVF embryos derived from high and low quality of oocytes.2. Identify the effect of overexpression of CPEB1 on the development of IVF embryos derived from high and low quality of oocytes.3. Identify the effect of overexpression of CPEB1 on the development of cloned embryos and their gene expression profile.
Project Methods
Specific Aim #1: Determine CPEB1 expression levels at different stages of IVF embryos derived from high and low quality of oocytes. BCB staining in bovine oocytes, IVM, and generation of IVF embryo. Bovine oocytes will be aspirated from slaughterhouse-collected ovaries and incubated in 26 µM BCB in PBS supplemented with 5% FBS for 90 min at 38.5°C. Then the cumulus-oocyte-complexes (COCs) will be selected based on their coloration (BCB+ COCs have a blue cytoplasm and BCB- COCs have a colorless cytoplasm) and placed into IVM medium for 21h at 38.5°C. IVF will be performed as described previously (Keim et al. 2023). Briefly, cryopreserved semen will be thawed in a 35°C water bath. Live sperm will be isolated using a 45%/90% percoll gradient and suspended at a concentration of 1x106/mL and incubated with matured COCs at 38.5°C with 5% CO2 for 6 h. Cumulus cells will be removed by pipetting in 0.3 mg/mL hyaluronidase and washed in PBS prior to in vitro culture. The IVF embryos will be cultured in 40 µl of culture medium ((Synthetic oviductal fluid) SOF) microdrops for 7 days, with the medium refreshed every 2 days. The grade of blastocysts will be recorded according to IETS manual (Manual of International Embryo Transfer Society, 4th edition) before collecting for q-PCR. q-PCR of BCB+ and BCB- oocytes and different stages of IVF embryos. Single denuded MII oocyte or IVF embryo at designed stages (2-cell, 8-cell, 16-cell and blastocyst stages) will be deposited into 5µl of 1× phosphate buffered saline (AMRESCO), supplemented with Ribonuclease inhibitor (0.5U/μl) (AMRESCO), snap frozen in liquid nitrogen, and stored at −80°C. Total RNA extraction will be performed using Zymo Quick-RNA Microprep Kit. Reverse transcription and specific target amplification (RT-STA) will be performed to each single oocyte or embryo to convert the RNA to cDNA and to enrich target-specific cDNA prior to qPCR. Cycling parameters will include a 5 min initial enzyme activation at 95°C, 35 denaturation/extension cycles (95°C for 15 s followed by 60°C for 60 s), and a 3 min final extension cycle at 60°C. The melting curve (Tm) will be examined for all assays to verify the quality of each amplicon.Specific Aim #2: Identify the effect of overexpression of CPEB1 on the development of IVF embryos derived from high and low quality of oocytes. In vitro transcription (IVT) of RNAs and polyadenylation. RNA was previously extracted from whole bovine ovaries using PureLink@ RNA Mini kit according to the manufacturer's instructions, and first stand cDNA was synthesized by Reverse Transcriptase PCR, and cloned into a pGEM®-T vector (Promega). RNA will be synthesized by linearizing the pGEM-CPEB1 vector with a restriction enzyme, NotI, with site downstream to the CPEB1 cDNA insert. Then, the linear plasmid will be purified by phenol-chloroform extraction as previously described (Sambrook and Russell, 2006) to eliminate protein and especially RNA contamination from the template DNA. The IVT reaction will be conducted by using a T7 RNA polymerase kit (mMESSAGE mMACHINE T7 Ultra kit (Thermo Fisher Scientific San Jose, CA, USA)) using the linearized vector and following the manufacturer's instructions. After 2 to 3 h of incubation at 37° C, the transcripts will be treated with 1 μl of TURBO DNAse I (ThermoFisher) and incubated 37° C for 15 min. The transcripts will then be polyadenylated by adding the tailing reagents (10 μl ATP solution (10 mM), 10 μl of 25 mM MnCl2, 20 μl 5X E-PAP Buffer, 36 μl nuclease-free water, and 4 μl of E-PAP) to the 20 μl mMESSAGE mMACHINE® T7 ultra reaction with additional of 30 min incubation at 37°C. Polyadenylation of the transcripts, estimated to be on average of 200 As, will be confirmed by denaturing agarose gel electrophoresis. The expression of CPEB1 will be identified by immunocytochemistry of cells transfected with the IVT RNA. Briefly, the bovine fibroblast cells will be transfected with 3 μg IVT RNA using an electroporation method. The cells will be fixed with 4% paraformaldehyde at 24 h after transfection. Immunofluorescent staining will be used to identify the expression of CPEB1 in the transfected cells (primary antibody: anti-CPEB1 antibody (Abcam) and fluorescence secondary antibody: Alexa Fluor™ 488. Human HeLa cells and non-transfected fibroblasts will be used as positive and negative controls, respectively. CPEB1 mRNA injection in IVF embryos. Production of IVF embryos will be done the same way as described in Specific Aim 1. After fertilization, the 1-cell stage IVF embryos will be washed in SOF medium and 5 pl of CPEB1 mRNA (100, 200, 300, 400, and 500 ng/μl) and same concentration of mCherry mRNA will be co-injected into the cytoplasm using piezo drill system. Embryos only injected with mCherry mRNA will be used as a control. Non-lysed IVF embryos will be then cultured in the 40 µl of SOF microdrops for 7 days, with the medium refreshed every 2 days. The presence of fluorescence in each embryo will be determined under a fluorescent microscope the 24 h after injection. IVF embryos without fluorescence will be excluded from following experiments. The optimal CPEB1 concentration determined by the highest blastocyst rate will be selected for injection in SCNT embryos in Specific Aim 3.Specific Aim #3: Identify the effect of overexpression of CPEB1 on the gene expression profiles and development of BCB+ cloned embryos. Somaticcell nuclear transfer (SCNT)and microinjection of CPEB1 mRNA in SCNT embryos. SCNT will be performed as described previously (Keim et al., 2023). Briefly, a single donor fibroblast will be injected into the perivitelline space of an enucleated oocyte. Fusion of the somatic cell with the enucleated oocyte will be performed in fusion medium using 1 DC electric pulse of 3.2kV/cm for 22 microseconds. Reconstructed embryos will be activated by exposure to 5 μM ionomycin for 4.5 min followed by a four-hour incubation in 2 mM 6-DMAP and 10 μg/mL cycloheximide. Following activation, the reconstructed embryos will be injected with CPEB1 mRNA and/or mCherry mRNA into the cytoplasm using a piezo drill system. The culture conditions and fluorescence assessment of SCNT embryos will be done as described in Specific Aim #2 above. Single cell RNA sequencing of SCNT embryos. For single cell dissociation, zona pellucida-free SCNT embryos at 2-, 8-, 16-cell and blastocyst stages will be incubated in a 1:1 solution of Trypsin (Sigma-Aldrich) and Accutase (Millipore) and are separated by mouth pipetting. Individual cells will be lysed and magnetic beads will be used to separate the intact nucleus from the RNA transcripts. The supernatant that separates from the intact nucleus of each single cell will be transferred to a PCR tube and RNA libraries will be prepared following the MATQ-seq protocol. External RNA Controls Consortium (ERCC) spike-ins from Ambion will be added to the supernatant of each cell to create a standard baseline measurement of RNA. Sequencing will be performed on the Illumina NextSeq in the Genomics Core at USU. For this aim, IVF embryos at matched stages will be used as control for sequencing.Data analysis and interpretation. The real-time PCR data in Specific Aim 1 will be analyzed by the 2-ΔΔCt method. In Specific Aim 2, cleavage and blastocyst formation of injected IVF and SCNT embryos will be assessed at Day 2 and 7, respectively. Cleavage and blastocyst rate results will be analyzed using a generalized linear model (Jamovi software). In Specific Aim 3, all post-sequencing data analysis, including trimming and mapping, as well as quantification of gene-expression levels, which are related to embryo development, pluripotency, metabolism, and apoptosis, will be completed by the USU bioinformatics group.