Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
ANIMAL SCIENCE
Non Technical Summary
This project will investigate the molecular mechanism that control the de-differentiation of a somatic - body - cell when it is fused with an enucleated egg. Otherwise known as somatic cell nuclear transfer (SCNT) or cloning, this process was described for the first time in 1997 (Roslin UK) in sheep and in 1998 in cows by our group. Unfortunately the efficiency of the procedure remained very low due to a poor understanding of the mechanims.We are proposing to use the zebrafish as a model to understand the reasons why most of the time the embryos fail to develop. Insights gain in this animal model will be later used in domestic animals.Having an affordable and reliable system to clone agriculturally important species will facilitate worldwide distribution of genetically superior animals with desirable characteristics, including but not limited to, meat and milk production.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
To test our hypotheses, we propose to look at the transcriptome and epigenome of the somatic cell, unfertilized MII oocytes and SCNT embryos.The specific aims of this proposal are:Aim 1. Characterization of lineage specific somatic cells with known reprogramming efficiencies at the transcriptomic and epigenetic level.Aim 2. Identification of oocyte mechanisms of erasure and reestablishment of epigenetic marks during reprogramming.Aim 3. Gain and loss of function studies to determine the influence of candidate epigenetic factors governing the processes of acquisition of cell plasticity and onset of differentiation in the context of SCNT.
Project Methods
Aim 1. Characterization of lineage specific somatic cells with known reprogramming efficiencies at the transcriptomic and epigenetic level.Methodology. Previous studies from our laboratory identified somatic cell lines with different susceptibilities to reprogramming mediated by SCNT (Figure 2). For our aim, we will select a cell line of high efficiency and one of low efficiency of reprograming (HER and LER). Both groups will be subjected to SCNT and the transcriptome of embryos from both selected lines will be determined at EGA (in zebrafish is called 50% epiboly occurring at 5 hours post fertilization). Embryos produced by natural fertilization (NF) will be used as control. For that, embryos generated from the 3 groups HER, LER and NF (20 embryos per condition) will be subjected to RNA sequencing (RNA-seq) individually by submitting the cDNA samples to BGI Hong Kong (Ilumina Highseq 2000, 91 bp reads, 2G clean data, pair ended). Bioinformatic analysis will be performed (in collaboration with Dr. Hasan Otu, U. of Nebraska) to identify differentially expressed regions among the three different groups. Additionally, we will perform another supervised bioinformatics analysis that will prioritize genes and pathways involved in epigenetic modifications. This will provide data of which genomic regions fail to be reprogrammed after SCNT in LER-derived embryos in comparison with embryos from HER and IVF after SCNT. In this manner, we will identify regions of the genome that are most and least resistant to reprogramming (MRR, LRR) (Chung et al., 2015; Matoba et al., 2014).To characterize these identified MRR and LRR regions, we will go back to the HER and LER somatic cells and analyze their transcriptome (methodology as before) to elucidate also the expression status of genes within the identified MRR and LRR regions in the somatic cells (transcriptome and bioinformatic analysis as before). We hypothesize that the epigenetic status of these MRR and LRR regions impacts the capacity of the cell to be reprogrammed. To address this, we will determine the epigenetic status of those MRR and LRR regions in somatic cells. For that, we will perform chromatin immunoprecipitation sequencing (ChIP-seq) focusing on histone modifications and variants known to have a role in cell reprogramming and differentiation: H3K9me3, H3K27me3, H3K4me2, H3K36me3, H3K4me1, H3K27ac and macroH2A(Canovas, Cibelli, & Ross, 2012; Gaspar-Maia et al., 2013; Matoba et al., 2014; ShanZhi-yan et al., 2014). We will also perform global DNA methylation analysis, to determine the DNA methylation status of these regions. After bioinformatic analysis (as above), we will obtain data of the epigenetic status of previously identified MRR and LRR regions that determine the capacity of the somatic cell to be reprogrammed. Identified epigenetic differences in MRR and LRR will be also assessed in SCNT embryos at the EGA stage.Aim 2. Identification of oocyte mechanisms of erasure and reestablishment of epigenetic marks during reprogramming.Methodology. We will compare the transcriptome of the two different cell sources, HER and LER somatic cells, prior to fusion to the oocyte (data already collected in aim 1) and compare with the transcriptome of unfertilized MII oocytes, and SCNT embryos at every other step of development, from the one cell until onset of gastrulation (70% epiboly). A total of 6 different embryonic states will be profiled, in two different cell lines, plus IVF controls. RNAseq will be performed in single embryos, 10 embryos per condition, plus 10 MII oocytes, a total of 190 RNAseq will be required.We will then perform a bioinformatic analysis to determine SCNT transcriptome kinetics, i.e. the utilization of the oocyte RNAs as well as remnants of somatic cell specific genes over time.In summary, Aim 1 focuses on changes that take place in the epigenome of the somatic cells at EGA and Aim 2 covers the effects of the oocyte during SCNT. Ultimately, we will have the most comprehensive list of novel candidate epigenetic marks, epigenetic modifiers and pathways implicated in erasing epigenetic memory and reestablishing the embryonic epigenome.Aim 3. Gain and loss of function studies to determine the influence of candidate epigenetic factors governing the processes of acquisition of cell plasticity and onset of differentiation in the context of SCNT.Methodology. Candidate genes and pathways will be subjected to overexpression, complete lack of function or partial downregulation using the recently developed CRISPR-Cas9 system optimized for zebrafish (Hwang et al., 2013) and intron sequence specific knock-in developed using CRISPR-Cas9 system has been able to introduce large DNA sequences (Li et al., 2015). For knocking-out (KO) strategies, embryos will be injected at 1 cell stage with mRNA of Cas9 nuclease and sequence-specific gRNAs recognizing the beginning of the coding sequence, producing small indels in this region, leading to gene KO with a high efficiency. Once reaching reproductive age, these targeted animals will be crossed with wt, and F1 generation will be screened (T7 endonuclease I assay and sequencing) for the presence of mutations. It is expected that 50 to 70% of F1 will have deletion in one allele of the gene. Lack or decreased gene expression will be checked by QPCR and in situ hybridization and western blot.If KO of the target genes proves to be lethal for the embryos, we will use the same strategy described below for overexpression of genes, but instead of driving the gene of interest, we will express shRNA in the oocytes for the target gene we want to knockdown.For overexpression experiments, the candidate gene will be inserted in an intergenic region without any functional characteristic. The gene of interest will be overexpressed by doxycycline-inducible promoter with the transactivator driven by the oocyte specific promoter: ZP2, anchored to the GFP reporter by a 2A sequence. F1 generation and screening will be performed as above.It is worth mentioned that we are currently working with CRISPr-Cas9 in our laboratory. So far we have been able to generate KO zebrafish for the tyrosine hydroxylase gene, generating albino fish. We are now working on generating zebrafish that are capable of expressing GFP in the oocyte by targeting the ZP2 gene. If successful, we will then design a strategy to modulate expression of genes in the oocytes.