Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration

Abstract

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), β-casein (Csn2), whey acidic protein (WAP), or β-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a “precise” integration approach, placing the human granulocyte–macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2–11 µg/ml hGMCSF). This was probably caused by the absence of the 3’-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30–40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2–0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals’ health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

TAD border deletion at theKitlocus causes tissue-specific ectopic activation of a neighboring gene

“…Mouse Kit Δ30k line was established from a chimeric animal generated after mouse embryonic stem cells (mESCs) injection as described earlier 67 . Briefly, mESCs were transfected with CRISPR/Cas9 plasmids aimed at a border region between Kit and Kdr TADs (see gRNA in Supplementary Table 2).…”

Site specific insertion of a transgene into the murine α‐casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk

“…This finding is consistent with published data. [ 24 ] Densitometric scanning of Coomassie stained gels confirmed α‐casein as the most abundant mouse milk protein at a concentration of 20 mg mL −1 . [ 21 ] This represents around 20% of total milk protein.…”

“…[ 14 ] Furthermore, transgene expression from within the α‐casein gene (reported by Smirnov and colleagues) was low despite the absence of the selection marker. [ 24 ] These data suggest that the presence of a selection marker gene is not a likely reason for the reduced A1L transgene expression from within the mouse α‐casein gene. 2.…”

“…The insertion of a transgene into an allele which contains multiple exons appears to be less conducive to transgene expression than integration into genes with fewer exons (β‐casein: 9 exons, or β‐lactoglobulin: 7 exons). The reduction of the number of exons by removing additional splice sites supports transgene expression [ 24 ] (Table S4). 3.…”

“…Secondly, a recent publication by Smirnov and colleagues used a similar approach to insert the coding region of the human granulocyte macrophage stimulating factor (hGMCSF) into the mouse α‐casein gene utilizing CRISPR‐Cas9 stimulated homologous recombination. [ 24 ] The hGMCSF gene was inserted into the second exon (as in our experiments) and, as an alternative, into the entire genomic space of α‐casein exons 1 through 33. The authors showed low mRNA expression for the insertion into the 2nd exon, but high mRNA expression (analogous to the endogenous α‐casein gene) when the entire α‐casein gene was replaced.…”