animal models with a human hematopoietic and immune system are now important tools for studying the human immune system as well as human - specific infections , such as hiv-1     [ 1 , 2 ] .
recent approaches have involved the use of genetically immunodeficient mice that have been reconstituted using human - derived hematopoietic stem cells ( hscs ) ( known as humanized mice ) .
several immunodeficient mice have been developed and nod / scid mice with nk defective genetically modified mice are mainly used for this approach     [ 35 ] , because they exhibit deficiencies in macrophage and dendritic cell function , and complement as well as t and b lymphocyte deficiencies     .
rag-2c   mice are also commonly used for establishing humanized mice     [ 7 , 8 ] , and it is known that the genetic background of rag-2c   mice is influenced by the efficiency of human cell engraftment .
jak3 is a non - receptor - type tyrosine kinase crucial for mediating signaling from the common -chain of cytokine receptors , and jak3-deficient mice and common -chain - deficient mice show the same phenotype     .
based on these findings , we generated rag-2jak3 mice with c57/bl6 and balb / c genetic backgrounds .
balb / c rag-2jak3 mice showed high efficiency of both human cd34 hematopoietic stem cell ( hsc ) and peripheral blood mononuclear cell ( pbmc ) transplantation compared with c57/bl6 rag-2jak3 mice .
our data show that the genetic background of the mice influences human cell engraftment and balb / c rag-2jak3 mice are becoming an alternative source of humanized mice .
c57/bl6 rag-2/jak3 double - deficient mice were established by crossing rag-2-deficient mice      ( obtained from the center for animal resources and development , kumamoto university , japan ) and jak3-deficient mice   ( kindly provided by dr .
balb / c rag-2-deficient ( rag-2 ) mice and balb / c jak3-deficient ( jak3 ) mice were established by crossing rag-2 mice or jak3 mice with the balb / c strain for 10 generations , respectively .
balb / c rag-2/jak3 double - deficient ( rag-2jak3 ) mice were established by crossing balb / c rag-2 mice and balb / c jak3 mice , and were housed and monitored in our animal research facility according to institutional guidelines .
all experimental procedures and protocols were approved by the institutional animal care and use committee at kumamoto university .
peripheral blood and umbilical cord blood samples were collected after obtaining written informed consent and approval from the ethics committee of kumamoto university faculty of medical and pharmaceutical sciences .
cd34 cells were isolated using the cd34 progenitor cell isolation kit ( miltenyi biotec , sunnyvale , ca ) and transplanted into the liver of irradiated ( 4.0  gy ) newborn mice . at 12 weeks after transplantation , peripheral blood was obtained and analyzed by flow cytometry .   peripheral blood mononuclear cells ( 2  10 cells ) were transplanted into 5.0  gy irradiated adult mice ( 810 weeks old ) as described . at 2 weeks after transplantation ,
mouse spleen cells were stained with dx5-fitc ( pan nk marker ) , mcd122 ( il-2r)-pe , mcd19-apc , and mcd3-pe / cy7 ( ebiosciences , san diego , ca ) to detect murine lymphocytes .
peripheral blood samples were obtained from the retro - orbital sinus of transplanted mice , and red blood cells were lysed with an ammonium chloride - potassium buffer and stained with antimouse cd45-apc / cy7 and antihuman cd45-pb ( biolegends , san diego , ca ) .
cells were washed and resuspended in pbs supplemented with 3% fcs , 0.02% nan3 , and 1  g / ml propidium iodide and were analyzed using lsr ii ( bd biosciences , san jose , ca ) .
k562 cells ( obtained from riken cell bank , tsukuba , japan ) were cultured in rpmi1640 medium ( sigma ) supplemented with 10% fbs , and were transplanted subcutaneously into the right flank of c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice .
the generated c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice survived and bred well under the specific pathogen - free conditions . to confirm the predicted immunophenotype of c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice , single - cell suspensions from spleen cells were labeled with fluorescent antibodies against mouse dx-5 ( pan nk marker ) , cd122 ( il-2r ) , cd3 ( t - cell marker ) , and cd19 ( b - cell marker ) . in contrast to wild - type mice
, no b ( cd20 positive ) and t ( cd3 positive ) lymphocytes or nk cells ( dx-5 and cd122 double - positive cells ) were detected in either c57/bl6 or balb / c rag-2jak3 mice ( figure 1 ) . to determine the engraftment efficiency of human hematopoietic stem cells ,
2  10 cord blood - derived cd34 cells from normal volunteers were injected intrahepatically into c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice as described     [ 5 , 7 ] .
levels of human cell engraftment were determined 12 weeks later ( figure 2(a ) ) . of the engrafted c57/bl6 rag-2jak3 mice , 18 of 28 mice had less than 0.1% of human cd45 cells , and only 2 had > 10% human cd45 cells in the peripheral blood ( 1.7  4.5% , n = 28 ) . in contrast ,
balb / c rag-2jak3 mice were highly engrafted and reconstituted with human cells ( 37.8  21.5% , n = 24 ) ( figure 2(b ) ) . to determine the engraftment efficiency of human primary lymphocyte populations , 2  10 pbmcs from normal volunteers were injected intraperitoneally into c57/bl6 rag-2jak3 mice and balb / c rag-2jak3 mice .
although human cells ( human cd45 cells ) were detected in c57/bl6 rag-2jak3 mice , the degree of engraftment was very low ( 1.7  4.5% , n = 13 ) .
in contrast , balb / c rag-2jak3 mice were highly engrafted with human cells ( 37.8  21.5% , n = 12 ) ( figure 3 ) .
subcutaneous administration of k562 cells resulted in solid tumor formation in both strains of rag-2jak3 mice .
the weight of tumors in both strains showed no significant difference ( c57/bl6 : 0.59  0.26  g , n = 7 , balb / c 0.63  0.33  g , n = 7 , p >
in this study , we established c57/bl6 and balb / c strains of rag-2jak3 mice , and showed that balb / c rag-2jak3 mice supported a high level of human lymphohematopoietic cell engraftment , whereas c57/bl6 rag-2jak3 mice achieved little engraftment .
interestingly , both strains supported the engraftment of human hematopoietic malignancy .   humanized mice with reconstituted human hematopoietic and immune cells are becoming a powerful tool for the investigation of human biological systems and for translational research .
nod / scid mice have been used for these purposes ; however , human hematopoietic stem cell engraftment is not sufficient and functionally mature lymphocytes are not developed in these mice , mostly due to residual nk activity in this mouse . to overcome these limitations ,
nod / scid mice were intercrossed with nk defective genetically modified mice , and several strains of mice suitable for humanized mice were established     [ 35 ] . among them , nod / scid/c ( nog and nsg ) mice are becoming the gold standard for generating humanized mice . recently
, spira gene polymorphism has been identified as a determinant influencing the efficacy of human hematopoietic stem cell engraftment into the nod / scid strain .
meanwhile , stroma cells of the balb / c strain have been shown to support human hematopoiesis to a lesser extent but not other strains ( c57/bl6 , c3h , icr ) , indicating that other mechanisms support human cell engraftment in immunodeficient mice than spira gene polymorphism .
in fact , manz and colleagues described the reconstitution of human acquired and innate immunity in balb / c rag-2c mice    .
although no study has compared reconstitution efficiency directly between nod / scid/c mice and balb / c rag-2c mice , the efficiency of human hematopoietic stem cell engraftment seems to be higher in nod background from the previous studies    [ 1 , 2 ] .
however , the efficiency of reconstitution in balb / c rag-2c mice is high enough for human immune and hematopoietic cell research ( figures 2 and 3 ) and balb / c background has several advantages compared with nod / scid background .  
mice with the scid mutation lack functional t and b lymphocytes resulting from the absence of gene recombination events in t - cell receptors ( tcrs ) and immunoglobulins ; however , they are known to show a leaky phenomenon in which functional t and b lymphocytes are produced with aging or ionized irradiation     [ 13 , 14 ] .
in addition , the prkdc gene disrupted by the scid mutation is expressed broadly and is involved in dna repair , while expressions of rag-1 and rag-2 are limited to hematopoietic cells and are involved only in dna recombination of t- and b - cell receptor genes , indicating that scid mice are more sensitive to radiation - induced dna damage than their rag-1 or rag-2 counterparts .
established radioresistant nod rag-1c   mice and showed that they support a similar level of human lymphohematopoietic cell engraftment     , which could overcome the disadvantages of the scid mutation .
the nod / scid strain has a predisposition to thymic lymphoma due to an endogenous ectopic provirus ( emv-30 )    .
nod / scid and nod / scid/2 m mice have a short mean life span for this reason    [ 6 , 17 ] , while nod / scid/c ( nog and nsg ) mice do not develop thymic lymphoma for an unknown reason     .
balb / c strain could overcome these disadvantages of nod / scid strain , although efficiency of human hematopoietic stem cell engraftment is not better than nod / scid strain .   as the strain background is known to affect human - cell engraftment and to function markedly in scid mice     [ 1 , 2 , 18 ] , we established and directly compared c57/bl6 and balb / c strains of rag-2jak3 mice .
rag-2jak3 mice showed the same phenotype as rag-2c mice lacking t , b , and nk cells      ( figure 1 ) .
the efficiency of human hematopoietic stem cell and pbmc engraftment in c57/bl6 rag-2jak3 mice was extremely poor , although balb / c mice and rag-2jak3 mice appeared to have similar levels as balb / c rag-2c mice ( figures 2 and 3 ) .
the breeding performance of balb / c rag-2c mice was the same as balb / c mice in our study .
in addition , balb / c rag-2c mice were relatively radioresistant compared with nod / scid strains .
thus , balb / c rag-2jak3 mice are a very useful strain of immunodeficient mice derived from animals with defined molecular lesions and have distinct advantages over nod / scid - based immunodeficient mice .
in conclusion , we established c57/bl6 and balb / c strains of rag-2jak3 mice and showed that balb / c rag-2jak3 mice enabled high human hematopoietic cell engraftment .
 immunodeficient mice are becoming invaluable tools in human stem cell and tumor research . in this study 
 , we generated rag-2/jak3 double - deficient ( rag-2/jak3/ ) mice with a c57/bl6 and balb / c genetic background and compared the human lymphohematopoietic cell engraftment rate . 
 human cord blood - derived cd34 + hematopoietic stem cells were successfully engrafted into balb / c rag-2/jak3/ mice ; however , the engraftment rate was far lower in c57/bl6 rag-2/jak3/ mice . 
 transplantation of human peripheral blood mononuclear cells resulted in the same tendency . 
 thus , a balb / c background offers superior engraftment capacity than a c57/bl6 background and provides an attractive model for human hematopoietic cell engraftment . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion