ABSTRACT
Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used on mammalian oocytes is mRNA knockdown by microinjection of small interfering RNA (siRNA), with non-specific siRNA injection used as a technical control. Here, we investigate whether and how the microinjection procedure itself affects the transcriptome of bovine oocytes. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes.
Subject(s)
Gene Expression Regulation, Developmental , Gene Knockdown Techniques/methods , Microinjections/adverse effects , RNA, Small Interfering/administration & dosage , Animals , Cattle , Female , Gene Knockdown Techniques/adverse effects , Oocytes/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/metabolism , RNA-Seq , Single-Cell Analysis , Transcriptome/geneticsABSTRACT
The ultimate outcome in genome-editing research stepped into unknown territories last month when two babies were brought into the world with clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) facilitated knockdown of chemokine receptor 5 (CCR5). An immediate outcry by the public and the scientific community followed, which is still ongoing with much apprehensions and criticism of the ethical and scientific aspects of the procedure and its effects on the future of genome editing needed in other stubborn inheritable diseases for which there is no cure at present. With the debate on the consequences of this particular receptor knockdown still going on and the after-shocks in the form of queries expected to continue for some time in the future, we enter the arena of this particular genome editing as first responders with concerns regarding the neurological aftermath of CCR5 knockout in the babies born.
