Introduction:

Vitamin D is critical for embryonic skeletal formation and long bone growth. Without vitamin D, the hypertrophic cell zone will expand because the cartilage fails to calcify leading to rickets. Protein disulfide isomerase A3 (PDIA3), also known as ERp60, ERp57, Grp58, and 1,25-MARRS, is a multifunctional protein that has been associated with rapid membrane-initiated signaling by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in several cell types. Knockdown of PDIA3 in osteoblast-like cell line MC3T3 showed that reduction of PDIA3 expression led to remarkable decrease of PKC activation with 1,25 (OH) 2VitD3 treatment as well as a decrease in expression of several osteogenic genes compared to wild type cells, which indicated the important role of PDIA3 in vitamin D-regulated cartilage and bone development. However, the mechanism involved still remains unclear. The fact motivate us to generate the PDIA3 knockout mice which will serve as a good model to further explore the physiological function of PDIA3 and to understand the mechanism involved in PDIA3-mediated 1,25VitD3 signal pathway.

To date, the major problems we are facing during the generation an ERp60 Knockout mice is the lack of a liable genotyping method to distinguish the Knockout mice from the heterozygous mice. No knockout mice available from the previous breeding along with very lower percentage of wild type mice which did not match to the Mendel’s rule of Segregation. Therefore, the object of my project is firstly to establish a new and reliable method for genotyping, Secondly, to confirm the ablation of PDIA3 at protein level. Finally, the third objective is to explore the structural and functional abnormality in the PDIA3 knockout mice.

Methods: 1. DNA extraction and purification: Mice tail clips were sliced into small pieces to increase the surface area and were placed in an Eppendorf tube with 300 uL Lysis Buffer and 30 ul protinease K for each tail clip. The Eppendorf tubes were placed in a 55°C water bath for 24 hours. After twenty four hours, the samples were boiled in water to denature the solution. From this, the DNA concentration was measured used the Nanodrop and from this, the amount of DNA top be used in PCR was calculated. For long PCR, crude DNA solution was further purified using the Qiagen LongTange PCR Kit . 2. Long PCR and sequencing: 1 uL Purified DNA was used as template. The long PCR was performed according to the instructions of the kit (Qiagen). Primers (A1, A2 , B1, and B2) were designed as shown in figure 1. The long PCR products were identified on 0.7% agarose gel. The purified PCR product was then subjected to sequencing. 3. Regular PCR 1 uL of crude DNA sample was used as template for PCR. Primers (C1, C2, E1 and E2) were designed as shown in figure 1. The PCR products were confirmed by electrophoresis on 5% PAGE gel. 4. Western blot Protein prepared from mice tail clips was subjected to SDS-PAGE and blot with anti-PDIA3 antibody according to lab protocol. 5. Histological analysis Femurs and hearts dissected from 33-week-old female Het and KO mice were fixed in 10% neutral formalin and embedded in paraffin. The cross-sections were stained by H&E, toluidine and trichrome respectively.

Results:
1. Identification of insertion site by long PCR and sequencing The Long PCR results showed the estimate length and location of the insertion site. Since the A1+A2 is about seven to eight kb and the primers themselves add up to about 1.3 kB, the estimated insertion site is 6 kB downstream from Intron 1. This calculation is seen in Figure 1 below. In order to find the exact site of the insertion, it was sequenced. The sequencing results seen in Figure 2 shows that the exact insertion site is at 6328 bp and the location of each primer on Intron 1. From this result, new primers were designed to further dinstinguish the insertion site sequence. The new primers designed were E1, E2, C1 and C2. To test that these primers indeed worked and were at the correct size if amplified, PCR was run using the primers as seen in Figure 3. From these sizes and combinations, a genotyping method was created to try to separate between Knockout and Heterozygous. [pic] Figure 1 Long PCR results

[pic] Figure 2 sequencing of PCR product

2. Establishment of genotyping system To test the genotypes, regular PCR was run and a gel was run to confirm the presence or absence of the specific band. If the vector is inserted in the correct place, the E1-C2 and C1-C2 will be no band and E1-E2 and C1-E2 will show a band as will neo. If the insertion of the vector is made in the wrong place, the E1-C2 and C1-C2 will show a band because the intron will be sequenced since there is no vector there. If there was no insertion E1-C2 and C1-C2 will be positive and E1-E2 ,C1-E2 as well as neo will be negative. For genotypes, the wild type will be positive for E1-C2 and C1-C2 but will be negative for E1-E2, C1-E2, and neo since wild type represent no insertion. The heterozygous will be positive for all of them and the knockout will be negative for E1-C2 and C1- C2 but positive for E1-E2, C1-E2, and neo. These combined form a table of the possible wild type, heterozygous and knockout genotypes as seen in Figure 3. This method allows the difference between the heterozygous genotype and knockout genotype to be seen.
[pic]
Fig. 3 Expectable genotyping results using new genotype system The genotyping shown in Figure 4A shows the presence of samples two; three and seven are Knockout based on the genotyping method at the DNA level because of the presence of a band in C1-E2 and neo, but the absence of a band in E1-C2. There were three heterozygous ones, samples 1,4,and 8. The gel shows a positive band for all three showing the heterozygous genotype. For the wild-type there is some discrepencies because there are two true wild type with C1-E2 and neo being negative and E1-C2 being positive, but there are two other sample 5 and 6 that are the same as above except they are positive for neo which are the wild type that have wrong insertion of the vector.
[pic]
Figure 4 Identification of Knockout mice at DNA (A) and protein level (B)

3. Identification of knockout mice at protein level by western blots So after the genotyping showed knockout mice at the DNA level, Western blots were performed on the thought knockout mice in order to see if the mice were knockout on the protein level. The western blot, shown in figure 4B, showed no knockout mice as seen in above though on the DNA level they were knockout. In each sample there was a band at the right size for the PDIA3 protein from the ERp60 gene.Since the Western Blot did not show the existence of the ERp60 gene at the protein level, Histology was performed to see if there were any abnormalities that could be seen between the Knockout Mice and the Heterozygous Mice in the heart muscle and the growth plate.

4. Histological evaluation of knockout mice Histological analysis was done on cross section of the growth plate, heart muscle, of known Knockout mice and heterozygous mice in order to see if there was a noticeable difference in the histology and to confirm the genotypes being different. The heterozygous mice were used as a comparison because since the genotyping yielded a low percentage of wild-type and this comparison is also done with the same sex mouse, there were not enough wild type or that were for sure wild type to use as a control for histology. Histology analysis results were shown in Figure 7, the disorganized and narrower growth plates were found in the knockout mice in comparison to heterozygous mice (Fig. 7A,B). H&E stained cross-section of heart showed the heart cavity of knockout mice was bigger than that of heterozygous mice (Fig. 7C). The heart fibers of knockout mice seemed thicker and disarrayed in comparison to those of heterozygous mice indicating the hypertrophic change. Moreover, fibers stained as blue by trichrome were present among the heart fibers of knockout mice indicating the fibrosis process (Fig.7D).

[pic][pic]

[pic] [pic]

Fig. 5 Histology analysis of cross section of growth plate and heart H&E stained (A) and Toluidine stained growth plate (B); H&E stained (C) and trichrome stained (D) heart muscle

Discussion: Currently, there was no reliable way to identify the genotype of the mice used to breed ERp60 knockout mice as well as the offspring of the mice breeded. By mixing two heterozygous mice together, the expected resulting genotypes would be 25% wild type, 50% heterozygous, and 25 % ERp60 Knockout mice. Genotyping of the first results showed unexpected results with from ten samples, four being wild type and three heterozygous and knockout respectively. From the total of the 47 newer samples, that were offspring of two heterozygous mice there was thirteen percent wild type, sixty six percent heterozygous and twenty-one percent knockout. From the genotyping method described above, the expected percentage of neo positive should be around seventy five percent, yet the results show 87 % neo, which is very high. The insertion site for the ERp60 trapping vector is not know, and even PCR based on the neo gene expression can only identify the wild type mice but not the Knockout or the heterozygous mice. There was very little percentage of wild type ERp60 mice and there is a possibility that the heterozygous breeded with heterozygous were not truly heterozygous leading to one of them possibly being a Knockout. This could cause the wild type percentage to be low. It suggested also that the neo marker on the vector might not be the best way to determine genotyping between wild type and heterozygous since it seems that with this vector, there can be multiple insertions and since this can not be detected from the present way of genotyping, maybe another signal would be helpful to see if the vector was inserted multiple times. From these Western blots, there is a possibility that since there were no knockout mice in existence after birth that maybe the ablation of the ERp60 gene was lethal and that all knockout mice would be dead in uterus. If ablation of ERp60 gene was lethal, the idea that all the knockout mice were sill alive showed that either the knockout mice were not really knockout or they died in uterus and were never even born or genotyped. Also the Western blot could have shown an ERp60 gene that is a fusion of two proteins that has a similar nature and molecular weight causing the band on the Western Blot and not on the SDS-PAGE Gel which could have helped keep the Knockout mice from dying. Multiple insertions could have caused the ERp60 protein band on the Western blot by having a similar size and weight causing the band to show up on Knockout mice on the protein level. Another possibility of the reason the Western Blot showed a band at the ERp60 protein level but was genotyped as a knockout at the DNA level is in the mechanism of the gene trapping. The gene trapping theoretically should truncate the gene and not allow the gene to function. It seems that maybe that even though the gene was truncated that the coding just jumped over the insertion so that the gene was still coded therefore still showing a band at the protein level even though the insertion happened. The histology results indicated the disorganization of growth plate as well as heart hypertrophy and fibrosis in knockout mice in comparison to heterozygous mice. Therefore, more obvious difference could be expected when wild type mice are used as control. However, these histological abnormal need be confirmed on the new breeding.

Conclusions: The insertion happening at Intron 1 of ERp60 gene did not function and cells can still express ERp60 protein even if at the DNA level the insertion is affecting the expression of the DNA fragments amplified by the PCR primers. So far, the current system of breeding has not created a real Knockout mouse on the protein level. There are possibilities that the Knockout mouse could not express the ERp60 protein and died in the uterus and that the ablation on the ERp60 gene is lethal before birth. There needs to be confirm by new sets of breeding so a knockout with a knockout mouse and heterozygous with a heterozygous, a wild type with a wild type and a knockout with a wild type. This breeding will allow for the correct percentages of wild type, heterozygous and Knockout to be investigated. Since the mice used for the breeding are certain to be each genotype, breeding will allow for non contaminated percentages. The genotyping method currently used above can differentiate between Heterozygous and Knockout genotype. The weight of each mouse as well as the length of the body and long bone length will be taken from the knockout and wild type mice respectively. Histology will be performed on the long leg bone, cartilage, heart, liver, spleen, and kidney in order to further compare any significant differences in knockout mice versus wild type mice.

-----------------------

Title: Generation of ERp60 Knockout Mice
Date of report: 12/12/2007
Investigator: Dr. Barbara D. Boyan
Co-investigator: Kimberly WU
Lab instructor: Dr. Yun WANG

Approval of Report

[pic]1>?[pic] ) - øü

¢
¤
!
*
|
†
ðìÜìÜìÔÏʺ¦’}’}’l’l’W’W’W’l’l)h~qh~q5?CJH*[?]PJ\?aJnH tH h~q5?CJPJ\?aJnH tH )h~qh~q5?CJPJ\?aJnH o([pic]tH &h~qh~q5?CJPJ Investigator

Lab instructor

Grade:

(D)

(C)

(B)

(A)

B

A