Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy.

Abnormal synaptic formation and signaling is one of the key molecular features of autism spectrum disorders (ASD). Cortactin binding protein 2 (CTTNBP2), an ASD-linked gene, is known to regulate the subcellular distribution of synaptic proteins, such as cortactin, thereby controlling dendritic spine formation and maintenance. However, it remains unclear how ASD-linked mutations of CTTNBP2 influence its function. Here, using cultured hippocampal neurons and knockin mouse models, we screen seven ASD-linked mutations in the short form of the Cttnbp2 gene and identify that M120I, R533* and D570Y mutations impair CTTNBP2 protein–protein interactions via divergent mechanisms to reduce dendritic spine density in neurons. R533* mutation impairs CTTNBP2 interaction with cortactin due to lack of the C-terminal proline-rich domain. Through an N–C terminal interaction, M120I mutation at the N-terminal region of CTTNBP2 also negatively influences cortactin interaction. D570Y mutation increases the association of CTTNBP2 with microtubule, resulting in a dendritic localization of CTTNBP2, consequently reducing the distribution of CTTNBP2 in dendritic spines and impairing the synaptic function of CTTNBP2. Finally, we generated heterozygous M120I knockin mice to mimic the genetic variation of patients and found they exhibit reduced social interaction. Our study elucidates that different ASD-linked mutations of CTTNBP2 result in diverse molecular deficits, but all have the similar consequence of synaptic impairment.

In the originally published version of this article, the DNA sequence reported in the Experimental Procedures for the PQR in Drosophila cells was the incorrect variant. The correct DNA sequence for the PQR in Drosophila cells is 5'-GGAAGCGGAGAAGGTCGTGGTAGT CTACTAACGTGTGGTGATGTAGAAGAAAATCCTGGACCT-3'.

Cell lines expressing foreign genes have been widely used to produce a variety of recombinant proteins. However, generating recombinant protein-expressing cell lines is usually a lengthy process and the resulting protein expression levels are often inconsistent. Here, we describe an efficient method for making stable cell lines expressing any recombinant protein of interest in a controllable and quantifiable manner. We integrate transgenes into specific genomic loci using CRISPR/Cas9 such that transgene expression is driven by endogenous promoters to ensure consistent and predictable expression of the recombinant protein. Expression levels can be predetermined by selecting promoters from genes with the desired level of expression. To quantify recombinant protein expression, a protein quantitation reporter (PQR) is incorporated between the endogenous and foreign genes. The PQR allows equimolar production of the endogenous protein, the recombinant protein, and a fluorescent reporter. As a result, expression levels of both the endogenous and recombinant proteins can be continuously monitored using fluorescence.