Embryonic gene expression profiling using microarray analysis

2009 ◽  
Vol 21 (1) ◽  
pp. 22 ◽  
Author(s):  
Sadie L. Marjani ◽  
Daniel Le Bourhis ◽  
Xavier Vignon ◽  
Yvan Heyman ◽  
Robin E. Everts ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiling ◽  
Nuclear Transfer ◽  
Lower Cost ◽  
Preimplantation Embryos ◽  
Microarray Technology ◽  
In Vitro Production ◽  
Vitro Production ◽  
Level Of Analysis

Microarray technology enables the interrogation of thousands of genes at one time and therefore a systems level of analysis. Recent advances in the amplification of RNA, genome sequencing and annotation, and the lower cost of developing microarrays or purchasing them commercially, have facilitated the analysis of single preimplantation embryos. The present review discusses the components of embryonic expression profiling and examines current research that has used microarrays to study the effects of in vitro production and nuclear transfer.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

Abnormal offspring following in vitro production of bovine preimplantation embryos: A field study

1998 ◽  
Vol 49 (5) ◽  
pp. 883-894 ◽  
Author(s):  
A.M van Wagtendonk-de Leeuw ◽  
B.J.G Aerts ◽  
J.H.G den Daas
Keyword(s):  
Field Study ◽  
Preimplantation Embryos ◽  
In Vitro Production ◽  
Vitro Production

A Patient-Derived Xenograft Model for Identifying Therapies and Defining Mechanisms of TSLP-Induced CRLF2 Signals in Ph-like B-ALL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2633-2633
Author(s):  
Olivia L Francis ◽  
Terry-Ann MIlford ◽  
Ineavely Baez ◽  
Jacqueline Coats ◽  
Christopher L. Morris ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Mtor Pathway ◽  
In Vivo Model ◽  
In Vitro Production ◽  
Pathway Activation ◽  
B Cell Precursors ◽  
Vitro Production

Abstract Philadelphia chromosome (Ph)-like B cell acute lymphoblastic leukemia (B-ALL) is a high-risk leukemia with a gene expression profile similar to BCR-ABL1+ B-ALL. Approximately 50% of all Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. The poor outcomes associated with CRLF2 B-ALL represent a major clinical challenge and an important component of pediatric cancer health disparities. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. Activating JAK mutations are associated with CRLF2 B-ALL, but overall data indicate that JAK mutations are present in 50% or less of CRLF2 B-ALL. Our data show that normal primary human bone marrow (BM) stromal cells express TSLP, suggesting that TSLP-induced CRLF2 signals could play a role in the initiation, maintenance and progression of CRLF2 B-ALL, particularly in cases without JAK mutations. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) to human TSLP and CRLF2. Further, using phospho flow cytometry we show that mouse TSLP is unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP, confirming the species specificity of mouse TSLP. These findings underscore the importance and challenge of developing in vivo systems that can model human TSLP-CRLF2 interactions for evaluating therapies and studying leukemogenesis of CRRLF2 B-ALL. To address this challenge we engineered patient-derived xenograft (PDX) mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (-T) mice were produced by transplanting with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not -T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in CRLF2 B-ALL cells. ELISA assays showed normal serum levels of hTSLP (12-32 pg/ml) in +T mice, while hTSLP was undetectable in -T mice. Since TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and -T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to -T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary leukemia cells. +T and -T PDX mice were produced using primary CRLF2 B-ALL cells. BM was harvested and whole genome microarray was performed on isolated CRLF2 B-ALL cells. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to -T PDX mice, confirming hTSLP activity in the +T PDX mice. Next we tested whether +T PDX mice provide an in vivo model of B-ALL that more closely mirrors patients than -T PDX mice. +T and -T PDX mice were generated from primary high risk B-ALL. RNAseq gene expression profiles from primary patient B-ALL cells were compared to those of the same patient sample expanded in +T and -T PDX mice. The gene expression pattern in +T mice was significantly closer to the primary patient sample than those from -T mice. The +T and -T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

2015 ◽  
Vol 67 (6) ◽  
pp. 1483-1491
Author(s):  
F.D. Oliveira ◽  
J.R. Sangalli ◽  
F.V. Meirelles ◽  
F. Perecin ◽  
P.P.C. Silva Filho ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Production Techniques ◽  
Morphological Differences ◽  
Vitro Production

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

199 EFFECT OF PRODUCTION SYSTEM AND BREED ON RELATIVE GENE EXPRESSION IN BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 198 ◽  
Author(s):  
S. Wohlres-Viana ◽  
M. C. Boite ◽  
M. M. Pereira ◽  
W. F. Sa ◽  
J. H. M. Viana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Production System ◽  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
In Vitro Production ◽  
System Effect ◽  
Vitro Production

Embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. Nevertheless, there are a few studies showing the effect of in vitro environment on embryos from different bovine subspecies, such as Gyr (Bos indicus) and Holstein (Bos taurus). The aim of this study was to evaluate the relative abundance of aquaporin 3 (AQP3) and ATPase-α1 (Na/K-ATPase alpha 1) transcripts in blastocysts produced in vivo or in vitro from Gyr and Holstein cattle. The production system effect (in vivo × in vitro) for Gyr cattle and the breed effect (Holstein × Gyr) for in vitro-produced embryos were evaluated. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA). The cDNA obtained were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed with REST software© using the pair wise fixed reallocation randomization Test. There was no difference (P > 0.05) in gene expression for AQP3 and ATPase-α1 between in vivo- and in vitro-produced Gyr embryos, although the results suggest that the APQ3 gene was down-regulated (0.81 ± 0.31) and the ATPase-α1 gene was up-regulated (1.20 ± 0.65) in embryos produced in vitro. For breed effect within in vitro production system, ATPase-α1 gene was down-regulated in Holstein (0.56 ± 0.30) when compared with Gyr embryos (P < 0.05). The same trend was observed for AQP3 (0.58 ± 0.25), but with no difference (P > 0.05). In conclusion, the data suggest that embryo production system does not interfere with the transcript amount of the genes studied for Gyr cattle; however, the in vitro production system may have different effects on gene expression according to embryo breed. Other genes should be evaluated for a better understanding of these differences. Financial support: CNPq, Fapemig.

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