Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias

Figures

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  Figure 1. Confirmation of gRNA efficiency later used for transfection and silencing of MTTP or SAR1B by CRISPR/Cas9. Amplification products detected on 1% E-gel EX agarose of genomic DNA extracted from Caco-2/TC7 cell line transfected with (A) gRNA1 targeting MTTP, (B) gRNA2 targeting MTTP, (C) gRNA1 targeting SAR1B, (D) gRNA2 targeting SAR1B and (E) gRNA targeting HPRT1, used as a control gene, before (B,D) the enzymatic digestion efficiency test and after (A,D) the enzymatic digestion efficiency test. This method detects locus-specific DNA double-strand breaks formation by direct polymerase chain reaction (PCR) amplification and endonuclease activity that specifically cuts at hetero-duplex mismatches. Only mutated DNA could be targeted and cut by the Digestion Enzyme, as evidenced by the presence of two bands, indicated by red arrows, with sizes congruent with the distance between Cas9 cutting sites.

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  Figure 2. Example of DNA sequences of the targeted region in the MTTP gene, obtained by the Sanger sequencing method. Cells were cultured in Petri dishes to reach confluence, and DNA was extracted from each transgenic clone and wild-type Caco-2/TC7 cell (WT), which was used as a control. (a) The targeted DNA sequence of wild-type cells in the MTTP gene shows a fully determined unique sequence. (b) The targeted DNA sequence in the MTTP gene in clone MTTP-KO1 shows overlapping disrupted sequences beginning at nucleotide 258.

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  Figure 3. Knocking-out confirmation of MTTP or SAR1B by SDS-PAGE (4–10%) and Western blot analysis. Cells were cultured in T25 flasks for 21 days. Total protein was extracted from each transgenic clone and wild-type Caco-2/TC7 cells (WT), which were used as a positive control. Fifty μg of extracts were resolved by 10% SDS/PAGE, and the blot was stained with monoclonal antibodies raised against MTP or Sar1b and β-actin to ensure equal lane loading. (a) Analysis of MTP levels in two clones transfected with MTTP-gRNA (first two bands) and two clones transfected with SAR1B-gRNA (last two bands), revealing no 97 kDa band for the two first clones while being detected in wild-type cells (positive control) and in clones transfected with SAR1B-gRNA, highlighting two clones without protein, named MTTP-KO1 and MTTP-KO2. (b) Analysis of Sar1b levels in 2 clones transfected with MTTP-gRNA (first two bands) and two clones transfected with SAR1B-gRNA (last two bands), revealing no 23 kDa band for the two last clones while being detected in wild-type cells (positive control) and in clones transfected with MTTP-gRNA, highlighting two clones without protein named SAR1B-KO1 and SAR1B-KO2.

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  Figure 4. Gene expression measured by RT-qPCR in Caco-2/TC7 cells following CRISPR/Cas9 knock-out. Cells were cultured on inserts for 21 days. (a) MTTP and (b) SAR1B mRNA expression determined by RT-qPCR in two clones lacking MTP and two clones lacking Sar1b proteins (as seen on Western blots). Wild-type cells’ mRNA levels were assigned a value of 1. and MTTP- or SAR1B-KO mRNA levels are expressed as relative expressions to wild-type cells. Data are expressed as means ± SEM (n = 10). Asterisks indicate a statistically significant difference in gene expression levels between control and KO clones (*** p < 0.0001).

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  Figure 5. Characterization of clone gene expression. Cells were cultured on inserts for 21 days. We studied genes involved in fatty acid transport (FATP4, FABP2), intracellular lipid re-synthesis (ACAT2, DGAT1, DGAT2, MGAT2), lipid droplet (PLIN2) or chylomicron (APOB) formation, cholesterol metabolism (ABCA1, ABCG8, NPC1L1, PGP, SCARB1) and oxidative stress management (CAT, SOD1) in (a) MTTP-KO1, (b) MTTP-KO2, (c) SAR1B-KO1, (d) SAR1B-KO2. Wild-type cells’ mRNA levels were assigned a value of 1, and clones’ mRNA levels were expressed as relative expressions to wild-type cells. Gene expression was measured by RT-qPCR in KO and wild-type Caco-2/TC7 cells. Data are expressed as means ± SEM (n = 10). Asterisks indicate a statistically significant difference in gene expression levels between control and KO clones (* p < 0.05, ** p < 0.001, *** p < 0.0001). White bars represent control cells; black bars represent considered clones.

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  Figure 6. Representative images of cytoplasmic lipid droplets were observed under inverted microscopy images. Cells were cultured in 24-well plates for 21 days and received a medium containing 16% FBS (instead of 8% FBS) for 24 h. Oil Red O staining (×400 magnification) was performed in (a) wild-type Caco-2/TC7 cells, (b) clone MTTP-KO1, (c) clone MTTP-KO2, (d) clone SAR1B-KO1 and (e) clone SAR1B-KO2.

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  Figure 7. Lipid accumulation in KO clones and control cells. Cells were cultured on inserts for 21 days. Quantification of lipid accumulation was assessed by the ratio of increases in (a) red surface area (Oil red O staining), (b) lipid droplets count per field and (c) lipid droplets size between cells incubated without micelles and cells incubated with micelles for 16 h. Data are expressed as means ± SEM (n = 20). Asterisks indicate a statistically significant difference between control and KO clones (* p < 0.05, ** p < 0.001, *** p < 0.0001).

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  Figure 8. (a) Cholesterol and (b) triglyceride basolateral secretion in MTTP and SAR1B KO Caco-2/TC7 cells compared with wild-type Caco-2/TC7 cells. Cells were cultured on inserts for 21 days and were incubated with mixed micelles for 16 h. Triglyceride and cholesterol levels in the basolateral medium were quantified by fluorimetric assays. Data are expressed as means ± SEM (n = 4). Asterisks indicate a statistically significant difference in cholesterol concentrations between control and KO clones (** p < 0.001, *** p < 0.0001).

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  Figure 9. Vitamin E and its basolateral-derivative content after (a) free α-tocopherol, (b) TAC or (c) TPGS acute micellar delivery. Cells were cultured on inserts for 21 days and were incubated with mixed micelles containing α-tocopherol or TAC or TPGS for 6 h. Alpha-tocopherol and its derivative levels were measured by HPLC after liquid–liquid extraction with hexane. Data are expressed as means ± SEM (n = 4). For (b,c), grey bars with crosses represent free α-tocopherol and white bars with dots represent TAC (b) or TPGS (c). For all figures, asterisks indicate a statistically significant difference in TAC concentrations between control and KO clones (*** p < 0.0001) and a hash (#) indicates a statistically significant difference in TAC or TPGS concentrations between control and KO clones (# p < 0.05).