Creation of the 5-hydroxytryptamine receptor 7 knockout rat as a tool for cardiovascular research

Using CRISPR-Cas9 technology, we created a 5-HT₇ receptor global knockout (KO) rat, on a Sprague-Dawley background, for use in cardiovascular physiology studies focused on blood pressure regulation. A stable line carrying indels in exons 1 and 2 of the rat Htr7 locus was established and validated. Surprisingly, 5-HT₇ receptor mRNA was still present in the KO rat. However, extensive cDNA and genomic sequencing of KO tissues confirmed an 11 bp deletion in exon 1 and 4 bp deletion in exon 2. The exon 1 deletion resulted in a frameshifted mRNA sequence coding for a nonfunctional protein. While the Htr1B locus was a potential off-target for the guide RNAs designed for exon 2 of Htr7, there were no off-target sequence changes at this locus in the originating founder. When the F2 generation of KO was compared with wild-type (WT) counterparts, neither the male nor female KO rats were different in body size, fat weights, or mass of organs (kidney, heart, and brain) important to blood pressure. Females were smaller in mass than their counterpart males. Clinical measures of plasma from nonfasted rats revealed largely similar values, comparing WT and KO, of glucose, blood urea nitrogen, creatinine, phosphate, calcium, and albumin to name a few. Loss of a functional 5-HT₇ receptor was validated by the complete loss of relaxation to the 5-HT_1/7 receptor agonist 5-carboxamidotryptamine in the isolated abdominal vena cava. This newly created 5-HT₇ receptor KO rat will be of use to investigate the importance of the 5-HT₇ receptor in blood pressure regulation.