Abstract
Non-homologous end joining (NHEJ) is a DNA repair pathway that is required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, core factors Ku70 and Ku80 form a heterodimer called Ku, which recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes can result in immunodeficient phenotypes, including severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we demonstrated that deletion of pro-apoptotic factor Trp53 rescues embryonic lethality in mice with combined deficiency of Xlf and Mri. Furthermore, these Xlf-/-Mri-/-Trp53+/- mice possessed reduced body weight, severely reduced mature B and T cell counts in the spleen and thymus, and accumulation of progenitor B cells in the bone marrow. Combined inactivation of Mri and Paxx results in live-born mice with modest B cell phenotype. In contrast, combined inactivation of Mri and Dna-pkcs results in embryonic lethality. Therefore, we conclude that MRI is functionally redundant with XLF during B and T lymphocyte development in vivo, and that Xlf-/-Mri-/-Trp53+/- mice possess a leaky SCID phenotype.
1. Introduction
Non-homologous end-joining (NHEJ) is a DNA repair pathway that recognizes, processes and ligates DNA double-stranded breaks (DSB) throughout the cell cycle. NHEJ is required for lymphocyte development; in particular, to repair DSBs induced by the recombination activating genes (RAG) 1 and 2 in developing B and T lymphocytes, and by activation-induced cytidine deaminase (AID) in mature B cells [1]. NHEJ is initiated when core subunits Ku70 and Ku80 (Ku) are recruited to the DSB sites. Ku, together with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), form the DNA-PK holoenzyme [2]. Subsequently, the nuclease Artemis is recruited to the DSB sites to process DNA hairpins and overhangs [3]. Finally, DNA ligase IV (LIG4), X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF) mediate DNA end ligation. The NHEJ complex is stabilized by a paralogue of XRCC4 and XLF (PAXX) and a modulator of retroviral infection (MRI) [4, 5].
Inactivation of Ku70, Ku80, Dna-pkcs or Artemis results in severe combined immunodeficiency (SCID) characterized by lack of mature B and T lymphocytes [2, 3, 6-8]. Deletion of both alleles of Xrcc4 [9] or Lig4 [10] results in late embryonic lethality in mice, which correlates with increased apoptosis in the central nervous system (CNS). Inactivation of Xlf (Cernunnos) only results in modest immunodeficiency in mice [11-13], while mice which lack Paxx [14-17] or Mri [5, 18] display no overt phenotype.
The mild phenotype observed in mice lacking XLF could be explained by functional redundancy between XLF and multiple DNA repair factors, including Ataxia telangiectasia mutated (ATM), histone H2AX [19], Mediator of DNA Damage Checkpoint 1 (MDC1) [20, 21], p53-binding protein 1 (53BP1) [17, 22], RAG2 [23], DNA-PKcs [20, 24, 25], PAXX [4, 14, 15, 20, 26-28] and MRI [5]. However, combined inactivation of Xlf and Paxx [4, 14, 15, 20], as well as Xlf and Mri [5], results in late embryonic lethality in mice, presenting a challenge to the study of B and T lymphocyte development in vivo. It has also been shown that both embryonic lethality and increased levels of CNS neuronal apoptosis in mice with deficiency in Lig4 [9, 10, 29, 30], Xrcc4 [9, 31], Xlf and Paxx [20], or Xlf and Dna-pkcs [24, 25] is p53-dependent.
In this study, we rescue synthetic lethality from Xlf and Mri by inactivating one or two alleles of Trp53. We also show that the resulting Xlf-/-Mri-/-Trp53+/- mice possess a leaky SCID phenotype with severely reduced mature B and T lymphocyte counts in the spleen, low mature T cell counts in the thymus, and accumulated progenitor B cells in bone marrow. Finally, we demonstrate that MRI functions in B and T lymphocyte development in vivo, and that its roles are compensated by XLF.
2. Materials and Methods
2.1. Mice
All experiments involving mice were performed according to the protocols approved by the Comparative Medicine Core Facility (CoMed) at the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Xlf+/- [11] and Dna-pkcs+/- [2] mice were imported from the laboratory of Professor Frederick W. Alt at Harvard Medical School. Trp53+/- mice [32] were imported from Jackson Laboratories. Paxx+/- [16] and Mri+/- [18] mice were generated by the Oksenych group and described previously.
2.2. Lymphocyte development
Lymphocyte populations were analyzed by flow cytometry as described previously [16, 18, 19, 22]. In summary, cells were isolated from the spleen, thymus, and femur of 5-7-week-old mice and treated with red blood cell lysis buffer Hybri-Max™ (Sigma Aldrich, St. Louis, MO, USA; #R7757). The cells were resuspended in PBS (Thermo Scientific, Basingstoke, UK; #BR0014G) containing 5% Fetal bovine serum, FCS (Sigma Life Science, St. Louis, Missouri, United States; #F7524), and counted using a Countess™ II Automated Cell Counter (Invitrogen, Carlsbad, CA, United States; #A27977). Then, the cell suspension was diluted with PBS to get a final cell concentration of 2.5 × 107 cells/mL. Finally, surface markers were labeled with fluorochrome-conjugated antibodies and the cell population was analyzed using flow cytometry.
2.3. Class switch recombination
Spleens were isolated from 5-7-week-old mice and stored in cold PBS. Splenocytes were obtained by mincing the spleens, and naïve B cells were negatively selected using an EasySep Isolation kit (Stemcell™, Cambridge, UK; #19854). Lipopolysaccharide (LPS; 40 μg/mL; Sigma Aldrich, St. Louis, MO, USA; #437627-5MG) and interleukin 4 (IL-4; 20 ng/mL; PeproTech, Stockholm, Sweden; #214-14) were used to induce CSR to IgG1. Expression of IgG1 was analyzed by flow cytometry.
2.4. Antibodies
The following antibodies were used for flow cytometry analysis: rat anti-CD4-PE-Cy7 (BD Pharmingen™, Allschwil, Switzerland, #552775; 1:100); rat anti-CD8-PE-Cy5 (BD Pharmingen™, Allschwil, Switzerland, #553034; 1:100); anti-CD19-PE-Cy7 (Biolegend, San Diego, CA, USA, #115520; 1:100); hamster anti-mouse anti-CD3-FITC (BD Pharmingen™, Allschwil, Switzerland, #561827; 1:100); rat anti-mouse anti-CD43-FITC (BD Pharmingen™, Allschwil, Switzerland, #561856; 1:100); rat anti-mouse anti-CD45R/B220-APC (BD Pharmingen™, Allschwil, Switzerland; #553092; 1:100); rat anti-mouse anti-IgM-PE-Cy7 (BD Pharmingen™, Allschwil, Switzerland, #552867; 1:100); rat anti-mouse IgG1-APC (BD Pharmingen™, Allschwil, Switzerland; #550874; 1:100). A LIVE/DEAD™ fixable violet dead cell stain kit (ThermoFisher Scientific, Waltham, MA, USA; #L34955; 1:1000) was used to identify dead cells.
2.5. Statistics
Statistical analyses were performed using one-way ANOVA, GraphPad Prism 8.0.1. 244 (San Diego, CA, USA). In all statistical tests, p<0.05 were taken to be significant (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
3. Results
3.1 Inactivation of Trp53 gene rescued embryonic lethality in mice lacking XLF and MRI
Combined inactivation of Xlf and Mri has previously been shown to result in synthetic lethality [5]. To generate Xlf-/-Mri-/-Trp53+/- mice, we intercrossed an Mri-/- strain [18] with an Xlf-/- Trp53+/- [20] strain. Next, we selected and intercrossed triple heterozygous (Xlf+/-Mri+/-Trp53+/-), and later, Xlf-/-Mri+/-Trp53+/- mice. With PCR screening, we identified Xlf-/-Mri-/-Trp53+/- (n=11), Xlf-/-Mri-/- Trp53-/- (n=2), and Xlf-/-Mri-/-Trp53+/+ (n=1) (Fig. 1) among the resulting offspring. Mice lacking both XLF and MRI possessed reduced weight (12 g on average, p<0.0001) when compared with gender- and age-matched WT (19 g), Xlf-/- (19 g) and Mri-/- (20 g) controls (Fig. 1A,B). We used these XLF/MRI-deficient mice to further characterize the development of B and T lymphocytes in vivo.
3.2. Leaky SCID in Xlf-/-Mri-/-Trp53+/- mice
To determine the roles of XLF and MRI in lymphocyte development in vivo, we isolated the thymus, spleen, and femur from Xlf-/-Mri-/-Trp53+/- mice, as well as from Xlf-/-, Mri-/-, Trp53+/- and WT controls. Combined deficiency for XLF and MRI resulted in a 3-fold reduction in thymus size (32 mg on average, p<0.0001) and a 9-fold reduction in thymocyte count (1.9×107, p<0.0001) when compared to single deficient or WT controls (Fig. 1C). Similarly, both average spleen weight (22 mg, p<0.0001) and splenocyte count (2.0×107, p<0.0001) in Xlf-/-Mri-/-Trp53+/- mice decreased approximately 4-5 fold when compared with WT and single deficient controls (Fig. 1D). We did not detect any direct influence of Trp53 genotype on lymphocyte development. The reduced number of splenocytes in XLF/MRI-deficient mice could be explained by decreased populations of B and T lymphocytes observed in the Xlf-/-Mri-/-Trp53+/- mice (Fig. 1E-G). Specifically, CD3+ T cells were reduced 4-fold (p<0.0001), while CD19+ B cells were reduced 20-fold (p<0.0001) when compared with single deficient and WT controls (Fig. 1E-G). Likewise, counts of CD4+, CD8+ and CD4+CD8+ T cells in the thymus, as well as counts of CD4+ and CD8+ T cells in the spleen, were all dramatically reduced when compared with single deficient and WT controls (about 4-fold, p<0.0001; Fig. 1E,G,H). From these observations, we conclude that XLF and MRI are functionally redundant during B and T lymphocytes development in mice.
3.3. Leaky SCID in mice lacking XLF and PAXX
Combined inactivation of XLF and PAXX has been shown to result in embryonic lethality in mice [4, 14, 15, 20]. To determine the impact of XLF and PAXX on B and T cell development in vivo, we rescued the synthetic lethality by inactivating one allele of Trp53, as described previously [20]. The resulting Xlf-/-Paxx-/-Trp53+/- mice possessed 30-to 40-fold reduced thymocyte count (4.0×106, p<0.0001) when compared to WT (1.3×108), Xlf-/- (1.4×108) and Paxx-/- (1.7×108) mice. This is reflected in decreased levels of double-positive CD4+CD8+ cells, as well as decreased levels of single-positive CD4+ and CD8+ T cells (Fig. 1,2). Spleen development was dramatically affected in mice lacking XLF and PAXX compared to WT and single-deficient controls, due to the lack of B cells and decreased T cell count (Fig. 1,2). When compared with the WT and single knockout controls, Xlf-/-Paxx-/-Trp53+/- mice had a 400-to 600-fold reduction in CD19+ B splenocyte count (0.1×106, p<0.0001) and a 70-to 90-fold reduction in CD3+ splenocyte count (to 0.3×106) (Fig. 1F-H and Fig. 2). From these results, we concluded that XLF and PAXX are functionally redundant during the B and T lymphocyte development in mice.
3.4. Early B cell development is abrogated in mice lacking XLF and MRI, or XLF and PAXX
Reduced counts and proportions of mature B lymphocytes in Xlf-/-Mri-/-Trp53+/- mice suggest a blockage in B cell development in the bone marrow. To investigate this further, we isolated the bone marrow cells from femora of mice lacking XLF, MRI or both XLF/MRI, and analyzed the proportions of B220+CD43+IgM-progenitor B cells and B220+CD43-IgM+ immature and mature B cells. We detected only background levels of B220+CD43-IgM+ B cells in bone marrows isolated from Xlf-/-Mri-/-Trp53+/- mice (Fig. 3A,B). However, these mice exhibited a 2-to 3-fold higher proportion of pro-B cells when compared with WT, Mri-/- and Xlf-/- controls (Fig. 3A,C). Similarly, Xlf-/-Paxx-/-Trp53+/- mice also possessed background levels of IgM+ B cells (p<0.0001; Fig. 3A,B) while having 3-to 4-fold higher proportion of pro-B cells when compared with WT, Paxx-/- and Xlf-/- controls (p<0.0001; Fig. 3A,C). Therefore, we concluded that B cell development is blocked at the pro-B cell stage of Xlf-/- Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice.
3.5. Paxx-/-Mri-/- mice possess a modest phenotype
Both PAXX and MRI are NHEJ factors that are functionally redundant with XLF in mice. Combined inactivation of Paxx and Xlf [4, 14, 15, 20], or Mri and Xlf ([5]; this study) results in synthetic lethality in mice, as well as in abrogated V(D)J recombination in vAbl pre-B cells [4, 5, 14, 15, 27]. To determine if Paxx genetically interacts with Mri, we intercrossed mice that are heterozygous or null for both genes (e.g., Paxx+/-Mri+/-). We found that resulting Paxx-/-Mri-/- mice are live-born, fertile, and are similar in size to WT littermates (17 g, p>0.9999) (Fig. 4A,B). Specifically, we observed that Paxx-/-Mri-/- mice had normal thymocyte and splenocyte counts. Furthermore, Paxx-/- Mri-/- mice underwent normal T cell development that was indistinguishable from the WT, Paxx-/-, and Mri-/- controls (Fig. 1G,H and 4C). However, Paxx-/-Mri-/- mice had a reduction in the CD19+ B cell number (Fig. 1F) when were compared to WT, Paxx-/- and Mri-/- controls (p<0.0025). Moreover, CD19+ B cell counts were similar in Paxx-/-Mri-/- and Xlf-/- mice (p>0.9270). Paxx inactivation did not affect Ig switch to IgG1 in MRI-deficient B cells (Fig. 4D,E). The quantity of IgG1+ cells after CSR stimulation was similar between Paxx-/-Mri-/- and Mri-/- naïve B cells (p>0.48), although both were lower than that of the WT control, both at 72 h and at 96 h (p<0.05). From this, we can conclude that there is a genetic interaction between Paxx and Mri in vivo, and it is only detected in B cells.
3.6. Synthetic lethality between Mri and Dna-pkcs in mice
Both MRI and DNA-PKcs are functionally redundant with XLF in mouse development [5, 24]. Combined inactivation of Paxx and Mri (this study), or Paxx and Dna-pkcs [20] genes results in live-born mice that are indistinguishable from single deficient controls. To determine if Mri genetically interacts with Dna-pkcs, we crossed Mri+/- and Dna-pkcs+/- mouse strains, then intercrossed the double-heterozygous and Mri-/-Dna-pkcs+/- strains (Fig. 5A). We identified 12 Mri-/-Dna-pkcs+/+ mice and 12 Mri-/-Dna-pkcs+/- mice, but no Mri-/-Dna-pkcs-/- mice (out of 6 expected). To determine if double-deficient Mri-/-Dna-pkcs-/- embryos are present at day E14.5, we intercrossed Mri-/-Dna-pkcs+/- mice and analyzed embryos (Fig. 5B). We identified two Mri-/-Dna-pkcs-/- embryos (63mg), which were about 40% lighter than Mri-/- embryos (108mg) (Fig. 5C,D). A Chi-Square test (χ2) was performed to determine if the embryonic distribution data fits the mendelian ratio of 1:2:1 that is expected from Mri-/-Dna-pkcs+/- parents. With DF=2 and χ2=1.8, the corresponding p-value lies within the range 0.25<p<0.5. This affirms that our data fit the expected 1:2:1 distribution and suggests that Mri-/-Dna-pkcs-/- is synthetic lethal. Therefore, we can conclude that there is genetic interaction between Mri and Dna-pkcs in vivo.
4. Discussion
Genetic inactivation of Xlf [11], Paxx [4, 14-16], or Mri [5, 18] in mice leads to development of modest or no detectable phenotype. However, inactivation of other NHEJ factors, such as Ku70-/- [6], Ku80-/- [7], Artemis-/- [3], Dna-pkcs-/- [2] results in blockage of B and T cell development in mice, while inactivation of Xrcc4 [9] and Lig4 [10] results in embryonic lethality. Moreover, combined inactivation of Xlf and Mri [5], Xlf and Paxx [4, 14, 15], or Xlf and Dna-pkcs [24, 25] also results in embryonic lethality, which is correlated with increased levels of neuronal apoptosis in the central nervous system (Fig. 6). Xlf also genetically interacts with Rag2 [23] and DDR factors, such as Atm, 53bp1, H2ax, and Mdc1 [17, 19-22, 33]. Xlf-/-Rag2c mice almost completely lack mature B cells and have significantly fewer mature T cells than single deficient controls [23]. Xlf-/-Atm-/- and Xlf-/-53bp1-/- mice are live-born and exhibit reduced body weight, increased genomic instability, and severe lymphocytopenia as a result of V(D)J recombination impairment in developing B and T cells [1, 17, 19, 22]. Xlf-/-H2ax-/- and Xlf-/-Mdc1-/-, on the other hand, are embryonic lethal [19-21]. There are several possible explanations for the functional redundancy observed between DNA repair genes. For instance, the two factors could have identical (e.g., if both proteins are involved in ligation or DNA end tethering) or complementary (e.g., if one protein stimulates ligation while the other is required for DNA end tethering) functions. To date, XLF has been shown to genetically interact with multiple DNA repair factors [1, 4, 5, 14, 15, 19, 20, 24, 25], and this list is likely to grow [33, 34]. However, no clear genetic interaction has been shown between Xlf and Artemis or Xrcc4 in the context of mouse development and V(D)J recombination [24], meaning that it remains difficult to predict genetic interactions without developing and characterizing genetic models.
Inactivation of one or two alleles of Trp53 rescues the embryonic lethality of Xrcc4-/- [9, 31], Lig4-/- [10, 30], Xlf-/-Dna-pkcs-/- and Xlf-/-Paxx-/- [20] mice (Fig. 6). Recent findings suggest that MRI forms heterogeneous complexes involving PAXX or XLF, which function during DNA DSB repair by NHEJ [5]. Additionally, combined inactivation of Xlf and Mri in vAbl pre-B cells results in a severe block in V(D)J recombination and accumulation of unrepaired DSBs in vitro, although it is unclear whether this combined inactivation will lead to a deficiency in B lymphocytes when translated to a mouse model [5]. Similarly, double deficient vAbl pre-B cells lacking Xlf and Paxx are also unable to sustain V(D)J recombination. Importantly, the lack of a progenitor T cell model system left the question of T cell development in Xlf-/-Mri-/- and Xlf-/-Paxx-/- mice completely unexplored. Inactivation of Trp53 resulted in live-born mice lacking XLF/PAXX [20]. These Xlf-/-Paxx-/-Trp53-/- mice had nearly no B and T cells, reduced size of spleen and hardly detectable thymus [20] (Fig. 6). Moreover, a conditional knockout mouse model, which results in double-deficiency of XLF/PAXX in early hematopoietic progenitor cells, was also able to overcome the embryonic lethality of Xlf-/-Paxx-/- mice [35]. With this model, impairment of V(D)J recombination in Xlf-/-Paxx-/- cells, as well as the resulting depletion of mature B cells and lack of a visible thymus could also be observed in vivo [35].
We have demonstrated that mice lacking XLF, MRI and p53, although live-born, possess a leaky SCID phenotype. Xlf-/-Mri-/-Trp53+/- mice have a clear fraction of mature B cells in the spleens (CD19+) and bone marrow (B220+CD43-IgM+) (Fig. 1,3,6), as well as clear fractions of double- and single-positive T cells in the thymus (CD4+CD8+, CD4+, CD8+) and single-positive T cells in the spleen (CD4+ and CD8+) (Fig. 1). However, the cell fractions from these mice are noticeably smaller than those of WT or single-deficient mice. Similarly, Xlf-/-Paxx-/-Trp53+/- mice are also live-born and possess a very small number of mature B cells in the spleen and bone marrow, as well as very minor fractions of single positive T cells in thymus and spleen (Fig. 2,3,6). Due to the smaller presence of mature B and T cells in these mice, we categorize the observed immunodeficient phenotypes as “leaky SCID”, which has previously been described in mice lacking other NHEJ factors, such as Ku70-/- [6], Artemis-/- [3], Lig4-/-Trp53-/-, Xrcc4-/-Trp53-/- [9, 31], Xlf-/-Atm-/- [19] and Xlf-/-Rag2-/- [23].
Both mature B and T cells are present in mice lacking XLF/PAXX and XLF/MRI. This can be explained by incomplete block in NHEJ and V(D)J recombination, in which the process is dramatically reduced but still possible. We also detected more mature T cells than B cells in these double-deficient mice. Potential explanations include longer lifespan of T cells, which accumulate over time following low efficiency of V(D)J recombination, while B cells are eliminated faster from the pool due to the different physiology [36, 37]. It is also possible that T cells we detected are a resultant subpopulation that is descended from the few cells that were able to bypass V(D)J recombination [12]. In this case, the repertoire of T cells based on T cell receptor in mice lacking XLF/PAXX and XLF/MRI would be significantly lower than in control mice, even if normalized to the total cell count.
It is important to note that inactivation of Trp53 is not always sufficient to rescue embryonic lethality in mice; for example, PLK1-interacting checkpoint helicase (PICH)-deficient mice possess developmental defects in the presence or absence of p53 [38], and ATR mutants (Seckel syndrome) are not completely rescued from embryonic lethality with the inactivation of Trp53 [39]. Embryonic lethality of XLF/PAXX and XLF/MRI double-deficient mice can be explained by the presence of Ku70/Ku80 heterodimer at the DSBs sites, which blocks DNA repair by alternative end-joining pathway(s), leading to massive apoptosis and cell cycle arrest [33]. Previously, it was shown that embryonic lethality of LIG4-deficient [40] and XLF/DNA-PKcs double-deficient mice [25] could be rescued by inactivating Ku70 or Ku80 genes. Similarly, we propose that inactivation of either Ku70 or Ku80 gene will rescue the embryonic lethality of XLF/PAXX and XLF/MRI double-deficient mice and will result in mice indistinguishable from Ku70- or Ku80-deficient controls.
We also found that mice with combined inactivation of Paxx and Mri (Paxx-/-Mri-/-) are live-born, fertile, and undergo almost normal B and T cell development (Fig. 4), where only the number of splenic B cells is affected, giving rise to a modest phenotype. Moreover, inactivation of Paxx did not affect the CSR efficiency in in vitro stimulated MRI-deficient B cells (Fig. 4), thereby confirming our observations in vitro. It has been shown that combined inactivation of Paxx and Mri genes in vAbl pre-B cells lead to similar V(D)J recombination efficiency to single deficient Mri-/-, Paxx-/- and WT controls [5]. Furthermore, combined inactivation of Paxx and Ku80 (Paxx-/-Ku80-/-), or Paxx and Atm (Paxx-/-Atm-/-) [15], as well as Paxx and Dna-pkcs (Paxx-/-Dna-pkcs-/-) [20] lead to a phenotype similar to their single deficient controls, Ku80-/-, Atm-/- and Dna-pkcs-/-, correspondingly. Thus, we conclude that there is a genetic interaction between Paxx and Mri.
Both Mri an Dna-pkcs genetically interact with Xlf. Strikingly, we found that combined inactivation of Mri and Dna-pkcs (Mri-/-Dna-pkcs-/-) leads to embryonic lethality, and that E14.5 Mri-/- Dna-pkcs-/- murine embryos were about 40% smaller than single-deficient siblings. DNA-PKcs is associated with the N-terminus of the MRI and Ku heterodimer in the process of recognizing DSBs [5], which may account for genetic interaction between Mri and Dna-pkcs. Thus, inactivation of Trp53, Ku70 or Ku80 may be a viable method to rescue synthetic lethality from Mri-/-Dna-pkcs-/- mice.
Xlf and Mri interact genetically, and mice lacking XLF/MRI are embryonic lethal. It was demonstrated by Hung et al. (2018) [5] previously, and largely confirmed in this study; however, we were able to identify one Xlf-/-Mri-/-Trp53+/+ mouse at day P30 post-birth. This mouse resembled Xlf-/- Mri-/-Trp53+/- and Xlf-/-Mri-/-Trp53-/- mice of similar age with respect of B and T cell development, although this mouse was generally sicker than its littermates and had to be euthanized (Fig. 1, 6). Similarly, one live-born Xlf-/-Paxx-/- mouse was reported by Balmus et al. (2016) [15], indicating that, exceptionally, embryonic lethality in NHEJ-deficient mice can be overcome, likely due to activity of alternative end-joining.
In conclusion, we have developed and described several complex genetic mouse models (Fig. 6). Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possessed severely impaired B and T lymphocyte development; Paxx-/-Mri-/- mice develop a modest B cell phenotype; and Mri-/-Dna-pkcs-/- mice are embryonic lethal.
Author contributions
VO, SCZ, QZ, AL and MFB designed the study, analyzed and interpreted the results. SCZ, QZ, AL and MFB performed most of the experiments. VO wrote the paper with the help of SCZ, QZ and RY. All the authors contributed to writing of the final manuscript.
Conflict of interest statement
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Research Council of Norway Young Talent Investigator grant (#249774) to V.O. In addition, VO group was supported by the Liaison Committee for Education, Research, and Innovation in Central Norway (#13477; #38811); the Norwegian Cancer Society (#182355); the Research Council of Norway FRIMEDBIO grants (#270491 and #291217), and The Outstanding Academic Fellow Program at NTNU (2017–2021).
Footnotes
Revised English/grammar. Added new data and statistics. Updated results, discussion and conclusions.