Sperm mediated human coagulation factor VIII gene transfer and expression in transgenic mice.
BACKGROUND
B-domain deleted human coagulation factor VIII cDNA (BDD-hFVIIIcDNA) transgenic mice were produced by using sperm mediated gene transfer (SMGT). The transcription and expression of human FVIII in transgenic mice were also investigated.


METHODS
Sperm were isolated from caudae epididymides of male C57BL/6 mice and transfected with linearized RC/RSV-BDDhFVIIIcDNA plasmid, and subsequently used to fertilize female mice via artificial insemination in vivo. After birth, F0 progeny were identified by PCR and Southern blotting for BDD-hFVIIIcDNA transgenic mice. F1 progeny were subsequently derived from a male transgenic F0 mouse and a normal C57BL/6 female mouse. The F1 progeny were then identified as BDD-hFVIIIcDNA transgenic mice by Southern blotting. The transcription and expression of BDDhFVIIIcDNA in transgenic mice were determined by northern blotting, western blotting and immunohistochemical staining. Blood was also collected from both F0 and F1 progeny to detect hFVIII:Ag and anti-hFVIII inhibitors.


RESULTS
A total of 9 F0 and 8 F1 progeny were delivered, in which 3 F0 and 2 F1 progeny were identified to have BDD-hFVIIIcDNA. The transcription and expression of BDD-hFVIIIcDNA were found to exist in the liver and kidneys of all transgenic mice. HFVIII:Ag in plasma of the transgenic F0 progeny was 31.95 ng/ml, 23.52 ng/ml and 26.36 ng/ml respectively, whilst the F1 transgenic mice showed results of 18.82 ng/ml and 12.16 ng/ml. Anti-hFVIII inhibitors were negative in both F0 and F1 progeny.


CONCLUSIONS
Human FVIII gene transgenic mice can be produced by the SMGT technique and express human FVIII protein in their bodies.
Yin
J
J
Division of Hematology, The Second Hospital affiliated to Medical College of Shantou University, Shantou, China. jyin@stu.edu.cn
Zhang
J J
JJ
Shi
G G
GG
Xie
S F
SF
Wang
X F
XF
Wang
H L
HL
D000818:Animals; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007315:Insemination, Artificial; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D013094:Spermatozoa; D014162:Transfection
C078147:F8 protein, human; D005169:Factor VIII