Figure S1.
Generation of eGFP-Col4a2 knock-in mice. (A) Targeting strategy for generating eGFP-Col4a2 knock-in mice. An eGFP protein was inserted into the N-terminus region of the 7S domain of the COL4A2 protein (between A28 and Q29). To minimize the structural interference of the inserted fluorescent protein with COL4A2 protein structure and functions, a glycine- and serine-rich flexible linker, GGSGGSGGSGGS, was added to both ends of the EGFP protein. L, linker; LF, left forward primer; LR, left reverse primer; RF, right forward primer; RR, right reverse primer. (B) Image of PCR screening used to distinguish wild-type, heterozygous, and homozygous eGFP-Col4a2 mice. Fabpi-200: An internal control primer pair for detecting the Fabpi gene. A 50 bp DNA ladder RTU (GeneDirex) was used. (C) Genome sequences of boundaries between the Col4a2 gene and linkers. (D) Immunofluorescence images of E13.5, E15.5, and E17.5 embryonic tissues from eGFP-Col4a2 mice stained for eGFP. Scale bars: 3 mm. (E–G) Immunofluorescence images of tissues from eGFP-Col4a2 mouse embryos. (E) At E15.5 (left panel), the eGFP (green) signal is detected at the epidermal BM (white closed arrowheads) below the keratin 14-positive (magenta) basal epidermis, the BMs of the hair placode (yellow closed arrowheads), the dermal vessel-like structures (open arrowheads), and the panniculus carnosus muscle-like layers (arrows). At E17.5 (middle and right panels), eGFP (green) is detected in perlecan-positive (magenta) epidermal and vessel-like BMs. (F) In the E15.5 kidney, eGFP (green) appeared around the tubular epithelial structures (closed arrowheads). (G) In the E15.5 brain, eGFP (green) appears in pial BM zones (closed arrowheads) and vessel capillaries (arrows). DAPI (blue) was used for nuclear counterstaining. Scale bars: (E and F) 20 μm, (G) 200 μm. (H–K) Immunofluorescence images of adult tissues from eGFP-Col4a2 mice. (H) In P56 adult telogen dorsal skin tissues from het mice, eGFP (green) colocalizes with collagen IV (magenta) and perlecan (magenta) in the BMs of the epidermis (closed white arrowheads), hair follicle epithelium (closed yellow arrowheads), arrector pili muscle (arrows), and vessel-like tissues (open arrowheads). (I) In P56 adult telogen dorsal skin tissues from homo mice, eGFP (green) is distributed as it is in het mice and colocalizes with perlecan (magenta). (J) In adult kidneys, eGFP (green) is detected in Bouman’s capsules (closed arrowheads), the mesangial matrix (arrows), and collecting ducts (open arrowheads). The same field of view is presented in Fig. 1 F. (K) In the subventricular zone, eGFP (green) colocalizes with perlecan (magenta) in capillaries (closed arrowheads) and fractone-like structures (open arrowheads) around GFAP+ (magenta) astrocytes. DAPI (blue) was used for nuclear counterstaining. Scale bars: 50 μm. Source data are available for this figure: SourceData FS1.

Generation of eGFP-Col4a2 knock-in mice. (A) Targeting strategy for generating eGFP-Col4a2 knock-in mice. An eGFP protein was inserted into the N-terminus region of the 7S domain of the COL4A2 protein (between A28 and Q29). To minimize the structural interference of the inserted fluorescent protein with COL4A2 protein structure and functions, a glycine- and serine-rich flexible linker, GGSGGSGGSGGS, was added to both ends of the EGFP protein. L, linker; LF, left forward primer; LR, left reverse primer; RF, right forward primer; RR, right reverse primer. (B) Image of PCR screening used to distinguish wild-type, heterozygous, and homozygous eGFP-Col4a2 mice. Fabpi-200: An internal control primer pair for detecting the Fabpi gene. A 50 bp DNA ladder RTU (GeneDirex) was used. (C) Genome sequences of boundaries between the Col4a2 gene and linkers. (D) Immunofluorescence images of E13.5, E15.5, and E17.5 embryonic tissues from eGFP-Col4a2 mice stained for eGFP. Scale bars: 3 mm. (E–G) Immunofluorescence images of tissues from eGFP-Col4a2 mouse embryos. (E) At E15.5 (left panel), the eGFP (green) signal is detected at the epidermal BM (white closed arrowheads) below the keratin 14-positive (magenta) basal epidermis, the BMs of the hair placode (yellow closed arrowheads), the dermal vessel-like structures (open arrowheads), and the panniculus carnosus muscle-like layers (arrows). At E17.5 (middle and right panels), eGFP (green) is detected in perlecan-positive (magenta) epidermal and vessel-like BMs. (F) In the E15.5 kidney, eGFP (green) appeared around the tubular epithelial structures (closed arrowheads). (G) In the E15.5 brain, eGFP (green) appears in pial BM zones (closed arrowheads) and vessel capillaries (arrows). DAPI (blue) was used for nuclear counterstaining. Scale bars: (E and F) 20 μm, (G) 200 μm. (H–K) Immunofluorescence images of adult tissues from eGFP-Col4a2 mice. (H) In P56 adult telogen dorsal skin tissues from het mice, eGFP (green) colocalizes with collagen IV (magenta) and perlecan (magenta) in the BMs of the epidermis (closed white arrowheads), hair follicle epithelium (closed yellow arrowheads), arrector pili muscle (arrows), and vessel-like tissues (open arrowheads). (I) In P56 adult telogen dorsal skin tissues from homo mice, eGFP (green) is distributed as it is in het mice and colocalizes with perlecan (magenta). (J) In adult kidneys, eGFP (green) is detected in Bouman’s capsules (closed arrowheads), the mesangial matrix (arrows), and collecting ducts (open arrowheads). The same field of view is presented in Fig. 1 F. (K) In the subventricular zone, eGFP (green) colocalizes with perlecan (magenta) in capillaries (closed arrowheads) and fractone-like structures (open arrowheads) around GFAP+ (magenta) astrocytes. DAPI (blue) was used for nuclear counterstaining. Scale bars: 50 μm. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal