TY - JOUR

T1 - Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration

AU - Smirnov, A. V.

AU - Kontsevaya, G. V.

AU - Shnaider, T. A.

AU - Yunusova, A. M.

AU - Feofanova, N. A.

AU - Gerlinskaya, L. A.

AU - Serova, I. A.

AU - Serov, O. L.

AU - Battulin, N. R.

N1 - Funding Information: This work was supported by Russian Science Foundation [Grant Number 16-14-00095]; Embryonic stem cell microinjection experiments performed at the Center for Genetic Resources of Laboratory Animals at ICG SB RAS that was supported by project #0259-2021-0016 from the Russian Government Budget. WGS data analysis were performed on a high-throughput computational cluster of Novosibirsk State University with support from the Ministry of Education and Science of Russian Federation, Grant #2019-0546 (FSUS-2020-0040). Publisher Copyright: © 2022, The Author(s).

PY - 2022/12

Y1 - 2022/12

N2 - 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.

AB - 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.

KW - Allergens/metabolism

KW - Animals

KW - Caseins/genetics

KW - Lactoglobulins/genetics

KW - Mammary Glands, Animal/metabolism

KW - Mice

KW - Milk/metabolism

KW - Milk Proteins/genetics

KW - Transgenes

UR - http://www.scopus.com/inward/record.url?scp=85130018549&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/16362cf9-78f2-39b8-803c-612548f27f58/

U2 - 10.1038/s41598-022-12071-1

DO - 10.1038/s41598-022-12071-1

M3 - Article

C2 - 35568783

AN - SCOPUS:85130018549

VL - 12

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 7983

ER -