Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies.
Previously we created two strains of factor VIII-deficient mice by insertion of a neo gene into (1) the 3' end of exon 16 and (2) exon 17 of the factor VIII gene. Affected mice of both strains have no plasma factor VIII activity, yet are healthy with no spontaneous bleeding. Factor VIII-deficient females bred with affected males survive pregnancy and delivery. We used reverse transcriptase-polymerase chain reaction of liver RNA to characterize factor VIII mRNA processing. Factor VIII mRNA of the exon 16 knockout strain contains neo sequences plus 17 bp of intron 16 due to use of a cryptic donor site in intron 16. All factor VIII mRNA of the exon 17 knockout strain lacks exon 17 and neo sequences. In skipping exon 17, the intron 16 donor site or a cryptic donor site 46 bp 3' to the intron 16 donor site are used. Thus, factor VIII deficiency in exon 16 knockout mice is due to truncated protein, while in exon 17 knockout mice it is due to either truncated or partially deleted protein. After immunizing exon 16 knockout mice with human recombinant factor VIII, two monoclonal antibodies were obtained that recognize < 100 pg of mouse factor VIII light chain. Assay of cryoprecipitate from the plasma of affected mice failed to show factor VIII light chain.
Bi
L
L
Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Sarkar
R
R
Naas
T
T
Lawler
A M
AM
Pain
J
J
Shumaker
S L
SL
Bedian
V
V
Kazazian
H H
HH
D000818:Animals; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D018390:Gene Targeting; D006467:Hemophilia A / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D005169:Factor VIII