The Sickle Cell Anemic Mouse

Paszty and sickle cell anemic mouse with only human hemoglobin; Lynn Yarris,, the new development towards a cure or treatment of sickle cell anemia has lead to the development of sickle cell anemic mice. From the late eighties two labs, Ryan et al. and Paszty et al., have been working on projects to breed and develop mice which have the same symptoms as humans with sickle cell anemia. These mice are the break through for sickle cell anemia. With the mice more testing can be done to examine the different treatment methods of sickle cell anemia as well as the development of the disease from onset.

Ryan et al. has worked on the construction of a mouse model for sickle cell anemic traits that could be bred and used for research purposes. Results from other researchers had shown that mice could be developed which could make high amounts of human alpha and beta-globin using the human hemoglobin genes inserted after the DNase I super-hypersensitive sites (also called LCR or locus control regions) injected into fertilized mouse eggs. Ryan et al. used the same strategy to create mice with human alpha -globin genes and human betaS or HbS globin. The mice that developed after this injection were examined for the inserted DNA in the correct orientation for transcription. Three progeny were found with head to tail tandem arrays of the transgenes, which produced HbS protein. The mouse that had the highest HbS expression was the initiator of a line of transgenic mice with HbS expression.
Testing the HbS mice by deoxygenating the blood with nitrogen gas showed no real effect of the human HbS gene in the mice. This result would be expected because the mouse beta -globins are still being produced and interacting with the HbS proteins to buffer against polymerization, akin to the sickle cell carrier who shows no sickle cell symptoms. (Ryan, 1990)
To overcome the effect of the mouse beta-globin interactions with the HbS the mice were bred with a beta-globin thalassemic (beta-thal) mouse line to reduce the mouse beta-globin (mbeta-globin) levels. The HbS/beta -thal mice produced were heterozygous for the transgenes and the beta-thal mutation. mRNA levels were examined at the key developmental stages to assure that the HbS was expressed at the correct stage of development. Developmental progress of the disease is especially important in the mice because treatments for sickle cell anemia will depend on those stages in humans. The closer one mimicís the condition in the experimental stage the closer one can estimate the validity of a treatment or cure.

mRNA was also quantitated to determine the amount of HbS being expressed verses the mbeta-globin. In both the HbS mice and the HbS/beta -thal mice there was 3 to 5 times as much mRNA for the HbS than the mbeta -globin mRNA indicating a higher human protein content than mouse protein. For the human and mouse alpha-globin the mRNA amounts were similar.

Unlike the HbS mice erythrocytes that showed almost no sickling traits approximately 90% of the HbS/beta -thal erythrocytes showed sickling when treated with nitrogen gas to deoxygenate. HbS/beta-thal mice also showed characteristic spleenic enlargements of 2 to 4 times wild type size but are healthy otherwise. (Ryan et al., 1990)

Ryan et al. and Greaves et al. both produced transgenic mice with 50% HbS which mimicked the human sickle cell trait, but not the debilitating disease itself. The sickle cell trait resemblance of the transgenic murine erythrosiod cells was due to the expression of mbeta-globin and malpha-globin in the mouse cells. Ciavatta et al. and Yang et al. both deleted the mouse beta -globin genes and Paszty et al. deleted the malpha-globin genes. These mice lines showed anemic conditions similar to human beta and alpha thalassemia conditions and could be bred with transgenic mice with only adult human hemoglobin production. (Ryan et al., 1997)

Viability of these transgenic mice was a concern because the mice would only have only human hemoglobin and it was known that human fetal globin is a major factor in the survival of sickle cell disease patients in utero. Human fetal hemoglobin (HbF) accounts for 70 to 90% of the hemoglobin at birth. Since HbF has been proven to help control sickling of cells in humans with sickle cell disease the worry was that the mice wouldnít have that protection in utero and would die before birth. A new construct for HbS globin was made with the HbF gene ahead of the HbS gene to assure the survival of the mice in utero by assuring the switch from HbS to HbS after birth (HbF/HbS). (Ryan et al., 1997)

Figure 1 from Ryan et al. showing A. The LCR and human hemoglobin cosmid constructs and B. Amounts of human (h) and mouse (m) hemoglobin produced in transgenic mice verses controls; Ryan et al., 1997: p874Mice were bred to be heterozygous for m beta -globin, m alpha-globin and HbS. These mice were crossbred to produce the lineage of mice homozygous for the alleles without m beta -globin and m alpha -globin. These double knockout HbF/HbS mice are similar to humans in that they have high HbF globin expression at birth (approx. 30 to 50%) and "synthesize no murine hemoglobin" (Ryan et al., 1997). These mice also persist to have a high but declining HbF expression for a full month before the full hemoglobin switch to HbS is completed. The figure to the left shows this switch clearly in the B portion where a control mouse, HbS3 adult (heterozygous for the beta and alpha mouse globin knowouts showing some mouse globin expression), the HbS3 newborn with expression of only human hemoglobin and a similar adult with only human adult hemoglobin beta and alpha being expressed.

90% of the mice survived for 2 to 9 months, were fertile and had many of the same symptoms of sickle cell anemic patients with the disease including: spleens 7 to 20 times enlarged (sometimes 10% of the weight of the mouse), in vivo pathologies affecting the vital organs, and congested livers. The tissues of the spleen, liver and kidney showed the characteristic blockages associated with sickled cells. Similar to human sickle cell patients, these blockages reduced blood flow and caused organ deformities and damage.
(Ryan et al., 1997)

Paszty et al. developed the mouse line with both murine alpha -globin genes deleted. This mouse line was bred with a line created by Ciavatta et al. that lacked mbeta -globin to get a true heterozygotic line which was not homologous for the mbeta-globin and m alpha -globin genes. This line was then interbred with a sickle cell anemic mouse line developed by injecting three different fragments of human DNA carrying the human alpha , beta and human fetal hemoglobin genes G gamma and A gamma-globin along with the mini Locus Control Region (LCR or the DNase I super-hypersensitive sites). Similar to Ryan et al., Paszty et al. believed that the A gammaand G gamma globin genes would help the transgenic mice to survive the gestation period due to their anti-sickling properties. Paszty et al. saw approximately 4 to 26% expression of the HbF at birth without continued expression to which they attribute the many mouse deaths at birth.The figure below shows the low expression (in Part B) of the human gamma-globin or human fetal globin and the resulting adult.Figure 1 from Paszty et al., 1997, A. Shows the high performance liquid chromotography of a wild type mouse fetus, a transgenic fetus with human sickle cell beta-globin , alpha-globin and gamma-globin . Figure 1 B shows HPLC for newborn sickle cell anemic mice and adult sickle cell mice.

Their mice are slightly beta -thalassemic but have "normal appearance, activity and fertility". They have found sickle cell anemia symptoms in their mice such as irreversibly sickled cells (ISC), anemia, increased rigidity of erythrocytes, and multiple organ damage. The ISC formed in vitro from the HbF/HbS mice developed by Paszty et al. have the classic sickle cell signs of decreased osmotic fragility, and increased dynamic rigidity which are measured by osmotic gradient ektacytometry. Multiple organ damage caused by the ISCís of the mice has also been characterized using mouse organ histopathology. Damage to the sickle cell mice kidney, lungs, heart, spleen and liver has been reported to be similar to damage seen in the same organisms in sickle cell anemic patients. The heart and kidney had twice the normal mass hypothetically due to increased function and the spleen increased weight 13 times due to the accumulation of blocked vessels and such related to ISCís. The mice lungs showed vascular congestion and artifacts were found in the livers of the sickle cell anemic mice. (Paszty et al., 1997).

Sickling Damage in the kidney of a Ryan lab transgenic mouse; Barinaga, 1997 : p 803Both the Paszty et al. and the Ryan et al. transgenic mice show the complete expression of only human hemoglobin. Though both are developed from the same beta and alpha mouse hemoglobin mutants differing results were achieved for the injected insertion of the human beta , alpha and gamma genes into the mice embryos. While Paszty et al. say that they had "many" mice die as a result of only 4 to 26% HbF globin expression at birth, Ryan et al. observed levels of 30 to 50% HbF globin expression and approximately 90% survival rate of the sickle cell anemic mice. These numbers clearly show that while Paszty et al. has produced a transgenic mouse Ryan et al.ís mice are more viable. Paszty et al. had more analysis of the different tissues damage rate, including heart and lung, and the osmolarity of the sickle cells themselves. While these differences in data and results are interesting the more important fact to consider is that both teams have developed transgenic mice that can be ""indispensable"" (Barinaga, 1997).

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