1. A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII
by Scot A. Fahs, Matthew T. Hille, Qizhen Shi, Hartmut Weiler, and Robert R. Montgomery
Blood
Volume 123(24):
June 12, 2014
©2014 by American Society of Hematology

2. Conditional F8-KO alleles.
Conditional F8-KO alleles. (A) Targeted F8 gene. (B) F8F allele. Excision of the neomycin resistance cassette by Flp recombinase produces the floxed (F8F) allele, which is expressed normally. (C) F8KO allele. Excision of exons 17/18 from the F8F allele by Cre recombinase produces a F8KO allele. (D) Alternative mRNA splicing of the F8KO allele. The predicted exon 16/19 splice [F8KO(16/19)] and an alternatively spliced transcript [F8KO(Alt)] in which 46 bp of intron 16 is retained are produced in approximately equal amounts. (E) Domain structure of normal FVIII protein. The position of exons 17/18 within the A3 domain is noted. (F) Predicted FVIII polypeptide encoded by F8KO(16/19) mRNA. (G) Predicted FVIII polypeptide encoded by F8KO(Alt) mRNA. Flp recombinase target (FRT) sites are represented as open triangles, and Cre recombinase target (loxP) sites as gray triangles. The positions of duplex genotyping primers P1, P2, and P3 and RT-PCR primers P4 and P5 are shown. aa, amino acid; Ex, exon.
Scot A. Fahs et al. Blood 2014;123:
©2014 by American Society of Hematology

3. Genotyping PCRs. WBCs or tail DNA PCR was used for routine genotyping.
Genotyping PCRs. WBCs or tail DNA PCR was used for routine genotyping. A Cre-positive (experimental) and Cre-negative (control) male (F8 F/y) and female (F8 F/F) are shown for each tissue-specific Cre model. (A) Duplex PCR of WBC DNA using a common forward primer, P1, in combination with 2 alternative reverse primers, P2 and P3. As depicted in Figure 1B-C, a 712-bp product is amplified from the F8F allele and a 462,bp product from the F8KO allele. (B) Singleplex PCR of WBC DNA using primers P1 and P2 amplifies only the 712-bp F8F allele-specific product. (C) Singleplex PCR of WBC DNA using primers P1 and P3 amplifies a 462 bp F8KO allele-specific product but can also amplify a 1683-bp product from the F8F allele. (D) A Cre-recombinase coding sequence–specific PCR of WBC DNA identifies Cre+ mice. No signal is seen for the Vav1-Cre transgene because it contains a codon-optimized iCre sequence, which is not recognized by the native Cre primers we used. (E) Duplex PCR analysis of tail DNA using primers P1, P2, and P3 detects both F8F and F8KO alleles, especially important for Vav1- and Tek-Cre mice, in which WBC DNA undergoes complete F8F→KO gene conversion.
Scot A. Fahs et al. Blood 2014;123:
©2014 by American Society of Hematology

4. Plasma FVIII activity.
Plasma FVIII activity. A chromogenic assay was used to measure FVIII activity in plasma from tail bleeds. Males and females are shown separately, revealing an apparent sex difference. F8F represents Cre−/− control mice that result from breeding F8F/y Cre+/− males of the various Cre stains with F8F/F females. Alb, Vav1, Cdh5(Mlia), Cdh5(Spe), and Tek represent experimental Cre+/− littermates. F8KO is the stably inherited exon 17/18-deleted strain. Hepatocyte-specific Alb-Cre has no effect on FVIII levels, whereas all endothelial Cre models result in reduced plasma FVIII, culminating with a severe hemophilic phenotype in the most efficient Tek-Cre model. *P ≤ .05, **P ≤ n.s., nonsignificant.
Scot A. Fahs et al. Blood 2014;123:
©2014 by American Society of Hematology

5. Analysis of tissue gDNA and mRNA.
Analysis of tissue gDNA and mRNA. Tissue samples were collected from males following whole-animal saline perfusion. DNA and total RNA were isolated from the same tissue sample and analyzed by allele-specific PCR. Except for the F8KO/y mouse, all animals inherited a F8F/y genotype, either without Cre or with coinheritance of a Cre transgene as indicated. (A) A total of 25 ng of gDNA was amplified using a duplex PCR that yields a 712-bp F8F allele-specific product and a 462-bp F8KO allele-specific product. Primer P1, P2, and P3 binding sites are shown in Figure 1B-C. (B) For RT-PCR, 100 ng of total RNA was reverse transcribed and then analyzed by PCR using primers P4 and P5, which span the exon 17/18 deletion cassette (as shown in Figure 1D). An 819-bp product is amplified from the F8F allele, representing normally spliced F8 mRNA. For the F8KO allele, in addition to the 407-bp product predicted for exon 16/19 mRNA splicing (F8KO(16/19)), a 453-bp product is also present due to alternative splicing (F8KO(Alt)). The “Standards” panels consist of PCR products amplified from defined mixtures of F8F/y and F8KO/y liver gDNA (panel A) or total RNA (panel B) at various ratios as indicated (F8F:F8KO). n.a. indicates a sample that was not analyzed.
Scot A. Fahs et al. Blood 2014;123:
©2014 by American Society of Hematology

6. Transplantation of WT bone marrow into FVIIInull mice.
Transplantation of WT bone marrow into FVIIInull mice. Unfractionated C57BL/6 BMNCs or BMMCs were transplanted into lethally irradiated FVIIInull recipients. (A) Plasma FVIII levels in BMT recipients. Tail blood samples were collected, and FVIII levels were determined by chromogenic assay. (B) Tail-clip survival test. The tail clipping test was performed at 15 weeks after BMT.
Scot A. Fahs et al. Blood 2014;123:
©2014 by American Society of Hematology