Question

In Figure 3 (panel a and c), genomic DNA from mouse tail was included in the analysis. What was the purpose of including these samples?
Art++ ArtP70/P70 Tail C Art+i+ Pre B ArtP70/P70 Pro B Pre B Pro B Tail -3.0 kb JB143 11111 GI JB1.2/1.31 JB1.4- JB1.5- JB1.6- -3.0 kb -2.0 JH1- JH2- -1.0 -0.3 JH3- DB1 to JB1 -3.0 kb -0.2 JH4 i DQ52 to JH JH1- -2.0 kb -1.0 JH2- DB2 to JB2 JH3- JB1.1- JB1.2/1.35 JB1.4- -2.0 kb -0.5 181.5- -10 UHAL -0.2 D. to JH JB1.6- -0.3 VB10 to JB1 JH1 JH2- -2.0 kb - 1.0 JB1.1- JB 1.2/1.35 -2.0 kb JH3- JB1.4- JB1.5- JH- JB1.6- V.Q52 to DJ -2.0 kb VB12 to JB1 JH1- JH2- JH3- 330 bp 330 bp JHE -1.0 Normalization Art*+ Rag - ArtP70/P70 Art- -0.5 JH4- C -0.2 304 bp V,7183 to DJ. TCR8 RS ends 330 bp 330 bp Normalization Normalization

Answer 1

In A:

It was done to study the impact of P70 mutation on levels of D to J and V to DJ rearrangements within the IgH and TCR-β loci. PCR amplification has been performed of the rearranging loci followed by Southern blotting.

In TCR-β locus, significant retention of the PCR product corresponding to unrearranged alleles in Artemis-P70 homozygous mice can be observed compared with controls.

A decreased but detectable level of PCR products for Dβ1 to Jβ1 and Dβ2 to Jβ2 region rearrangements in ArtP70/P70 can also be seen.

With Vβ10- and Vβ12-specific PCR primers there is significantly reduction in the level of Vβ to DJβ1 rearrangements in the homozygous mutant thymocytes compared with controls.

It all indicates that there is significant decreases the frequency of both D to J and V to DJ rearrangements within the TCR-β locus because of Artemis- P70 hypomorphic mutation, yet substantial levels of recombination do occur.

In C:

DH to JH rearrangements is studied in pro- and pre–B cells from ArtP70/P70 and control mice using the same PCR approach. 5′ of DHQ52 paired with a primer 3′ of JH4 has been used for amplification.

A reproducible decrease in levels of DH to JH rearrangements in Artemis-P70 pro- and pre–B cells can be observed; however, significant levels of DH-JH rearrangements can also be detected.

VH to DJH rearrangements involving two VH families, VHQ52 and VH7183, are also reduced, but not abrogated, in ArtP70/P70 developing B cells.

The results indicates that in endegenous V(D)J recombination, the partial B and T immunodeficiency caused by Artemis-P70 is the result of an impairment. Thus, we can conclude that the mutant Artemis nuclease plays important role in in-vivo V(D)J recombination, but it is less efficient compared with the WT protein.

The purpose of including these samples is to study the effect of mutant Artemis nuclease in V(D)J recombination.