Freitag, Juli 29, 2022
StartBiotechnologyAnalysis of the α-casein (CSN1S1) locus as a possible goal for a...

Analysis of the α-casein (CSN1S1) locus as a possible goal for a site-specific transgene integration


Building of the donor vectors and modifications of mESCs

Donor vectors for insertion/substitute modifications had been based mostly on the OCT4-eGFP-PGK-Puro vector (Addgene #31937). PGK-Puro cassette was eliminated to exclude undesired results on the native regulation of the Csn1s1 gene after integration. Homology arms had been inserted consequently with Gibson cloning. Csn1s1 homology areas for insertion vector had been: Chr5: 87,814,490–87,815,105 (616 bp) (5′-arm) and Chr5: 87,815,135–87,816,185 (1051 bp) (3′-arm); for substitute: Chr5: 87,814,490–87,815,105 (616 bp) (5′-arm) and Chr5: 87,828,822–87,829,814 (993 bp) (3′-arm).

Mouse embryonic stem cells (mESCs, DGES1 cell line, 129S2/SvPasCrl) had been transfected with corresponding donor vectors (insertion/substitute), PuroR co-selection plasmid, Cas9 (Addgene #41815) and gRNA-expressing plasmids (Addgene #41824, spine) utilizing Lipofectamine 2000. Sequences of gRNAs are supplied in Sup. Figs. 5 and 6. Number of the constructive clones was carried out utilizing 2 μg/ml puromycin for two days. All cell strains had been grown at 37 °C with 5% CO2 and passaged each 2–3 days. Surviving ES colonies had been subcloned and PCR genotyped utilizing primers from Sup. Desk 4. PCR-positive clones had been examined by cytogenetic screening to pick these with regular karyotypes > 60% (10–20 metaphases per ES clone). Two chosen clones with hGMCSF integration (INS-GM and REP-GM) had been moreover analyzed with Sanger sequencing of homology arms, hGMCSF ORF and high off-target websites earlier than the technology of chimeric animals.

The embryonic stem cell strains used on this work can be found at a Collective Heart of ICG SB RAS Assortment of Pluripotent Human and Mammalian Cell Cultures for Organic and Biomedical Analysis (https://ckp.icgen.ru/cells/; http://www.biores.cytogen.ru/brc_cells/collections/ICG_SB_RAS_CELL).

Producing transgenic mouse strains through chimeric animals

Chosen ES clones had been plated on gelatin and cultured for 3 passages after thawing in commonplace ES cell 2i medium (1 μM PD, 3 μM CHIR, DMEM (ThermoFisher), 7.5% ES FBS (Gibco), 7.5% KSR (Gibco), 1 mM l-glutamine (Sigma), NEAA (Gibco), 0.1 mM β‐mercaptoethanol, LIF (1000 U/ml, Polygen), penicillin/streptomycin (100 U ml—1 every)).

CD-1 or B6D2F1 blastocysts from the superovulated females had been collected at day 3.5 put up coitus, washed out with M2 medium (Sigma-Aldrich) and positioned in KSOR medium (CosmoBio) lined with mineral oil at 37 °C, and injected with 10–15 cells below a microscope (TransferTip, Eppendorf). Blastocysts had been monitored in KSOM medium for two h at 37 °C, 5% CO2. Surviving blastocysts had been transplanted to pseudopregnant CD-1 females (12–13 per feminine). F0 males with a excessive share of chimerism had been crossed with CD1 or C57BL/6J females (Sup. Desk 3, 4). Agouti-colored F1 pups had been genotyped with primers for the transgene’s 5′-homology arm (Sup. Fig. 1). F1 animals had been crossed to provide F2 homozygous females for milk evaluation. To discriminate between hemizygous and homozygous hGMCSF inserts, primers for transgene’s 5′-homology arm and for mouse Csn1s1 had been used (Sup. Fig. 1, Sup. Desk 4).

Animals had been saved in a regular atmosphere at 24 °C temperature, 40–50% relative air humidity and 14 h mild/10 h darkish–light-cycle. Meals and water had been accessible advert libitum. Lactating mice had been euthanized by isoflurane inhalation instantly after milk assortment. On the finish of experiments, remaining animals had been euthanized by CO2. All experiments had been performed on the Centre for Genetic Assets of Laboratory Animals on the Institute of Cytology and Genetics, SB RAS (RFMEFI61914X0005 and RFMEFI61914X0010). All experiments had been carried out in accordance with protocols and pointers authorised by the Animal Care and Use Committee Federal Analysis Centre of the Institute of Cytology and Genetics, SB RAS working below requirements set by rules paperwork Federal Well being Ministry (2010/708n/RF), NRC and FELASA suggestions. Experimental protocols and euthanasia procedures had been authorised by the Bioethics Evaluation Committee of the Institute of Cytology and Genetics. The manuscript adopted the suggestions within the ARRIVE pointers.

Milk assortment and evaluation

Milk was collected with a pipette from narcotized feminine mice at day 10 of lactation after oxytocin administration and saved at − 80 °C. For ELISA evaluation, milk was diluted 1–6 million occasions with water and assayed in accordance with the producer’s advice (Human GM-CSF Quantikine ELISA Equipment, DGM00, R&D Programs). Blood was collected from the euthanized mice and the serum was diluted × 1000 occasions for the assay. Measurements had been taken at 490 nm with BioTek Epoch Spectrophotometer.

The protein concentrations in milk had been quantified utilizing Pierce BCA Protein Assay Equipment (ThermoFisher). For Western blot, equal quantities (20 μg) of milk samples had been separated on 15% SDS-PAGE, after which transferred onto Immun-Blot PVDF membrane (Bio-Rad). Membrane was blocked with 5% milk/TBST(20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for two h and incubated with major antibodies towards hGMCSF 1:500 at 4 °C in a single day (R&D, catalogue # AF-215-NA). Subsequent day, membranes had been incubated with horseradish peroxidase-conjugated secondary antibodies 1:1000 (#93702, Cell Signaling) for two h at 25 °C. Detection was carried out with SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580, ThermoFisher) and Chemidoc XRS Imaging system (Bio-Rad). Reversible staining of the PVDF membrane (Fig. 3A) was carried out with Pierce Reversible Protein Stain Equipment (#24585, ThermoFisher).

RNA extraction and ddPCR evaluation of expression

Whole mobile RNA was extracted from mouse mammary glands and different organs in glass homogenizers utilizing TRI Reagent (Sigma-Aldrich). 1 μg of whole RNA was used to generate cDNA in a 20 μl response utilizing RevertAid RT Equipment (Thermo Fisher Scientific) with random hexamer primers in accordance with the producer’s directions. Droplet digital PCR (ddPCR) was carried out utilizing a QX100 system (Bio-Rad) with primers and probes particular for the Csn1s1, Csn2, Rpl4, hGMCSF, and hGMCSF/Csn1s1 chimeric transcripts. The primers and probes sequences are offered in Sup. Desk 4. ddPCR reactions had been set in 20 μl volumes containing 1× ddPCR Supermix for Probes (no dUTP), 900 nM primers and 250 nM probes, and 1 μl of 20- or 3000-fold diluted cDNA or 20 ng DNA. ddPCR reactions for every pattern had been carried out in duplicates. PCR was performed in accordance with the next program: 95 °C for 10 min, then 41 cycles of 95 °C for 30 s and 61 °C for 1 min, with a ramp charge of two °C per second, and a closing step at 98 °C for five min. The outcomes had been analyzed utilizing QuantaSoft software program (Bio-Rad). Thresholds had been set to 6000 for the Csn1s1, Csn2, Rpl4, hGMCSFxCsn1s1 genes (Sup. Fig. 4); 4500 for the hGMCSF gene (Sup. Fig. 4B); 5000 for the Emid1 and hGMCSF DNA areas (Sup. Fig. 2B).

Immunohistochemistry

Intracardiac perfusion of euthanized mice was carried out manually with 4% PFA resolution in 1× PBS. Organs had been collected in 1× PBS and stuck in 4% PFA resolution in a single day on a curler shaker at 4 °C. Subsequent day samples had been washed in 1× PBS resolution thrice for 30 min. For tissue dehydration organs had been sequentially incubated for at the very least 24 h with a 15% and 30% sucrose resolution in 1× PBS at 4 °C. Subsequent, organs had been embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek) and frozen. Organ sections of fifty um thickness had been ready on MICROM HM 505N cryostat (Microm) and instantly collected on SuperFrost™ slides (Thermo Scientific). Sections had been washed with 1× PBS and incubated in blocking resolution: 2% BSA (Sigma Aldrich), 0.2% Triton X-100 (Amresco), 5% FBS (Capricorn Scientific). Major antibodies towards human GMCSF (R&D, catalogue # AF-215-NA) had been diluted in blocking resolution and incubated with sections in a single day at sluggish velocity on an orbital shaker at room temperature. Subsequent, slices had been washed with 1× PBS thrice for 20 min and stained with secondary antibodies (Jackson Immuno Analysis, catalogue # 705-165-147) and DAPI diluted in 1× PBS for two h at room temperature. Slices had been washed with 1× PBS thrice for 20 min and utterly dried out and at last had been mounted with ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific). Immunofluorescence was visualized below confocal fluorescence microscope LSM 780 NLO (Zeiss) with ZEN software program (Zeiss).

Complete genome sequencing

Genomic DNA was extracted from the tail tip of a homozygous REP-GM animal and subjected to paired-end 2 × 150 bp Illumina sequencing, producing roughly 317 million reads. Uncooked reads had been analyzed utilizing FastQC software program to make sure excessive knowledge high quality and mapped to the mouse genome (mm10) with Bowtie 2 with default parameters. To carry out knowledge visualization, alignments had been transformed to BAM format, sorted and listed utilizing SAMtools, and loaded because the IGV browser observe. For off-target mutations evaluation, chosen off-target websites (Sup. Fig. 5, 6) had been manually inspected within the IGV browser.

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