Engineering Mouse Lines Expressing Human Mutant ELA2 Using Bacterial Artificial Chromosomes

David Jin¹, Matthew Christopher and Daniel Link², Biology Department, Washington University, St. Louis, MO¹, Washington University School of Medicine

Severe congenital neutropenia (SCN), also known as Kostmann’s Syndrome, is a rare illness present from birth. Only about one in one hundred thousand people have this disease; however, its symptoms are severe and long lasting. Patients usually suffer from opportunistic infections, a significant risk of developing acute myeloid leukemia (AML), and a low neutrophil count (<200 cells/mm3), thus severely inhibiting a primary immune defense mechanism.

SCN is characterized by a block in myeloid maturation after the promyelocyte stage, leading to a sharp decrease in neutrophil formation. Recently, the pathogenesis of SCN has been attributed to heterozygous mutations of the ELA2 gene coding for neutrophil elastase (chromosome 19p13.3). To date, 27 different mutations causing 25 different amino acid changes have been found.

So far, only one animal model has been created to simulate this disease in humans. Dave Grenda et. al. generated a transgenic mouse model expressing a V72M mutation of their ela2, simulating a mutation found in two unrelated families with SCN. The mice showed a normal phenotype with healthy neutrophil numbers and no signs of developing AML. This could be the result of different structural and functional consequences of the V72M mutation on human and murine neutrophil elastase, different cellular environments and target proteins, or simply that the mutation may not be sufficient to cause SCN.

Since heterozygous germ-line mutants exhibit a mutant phenotype, these mutations seem to have a gain-of-function. We have hypothesized that the expression of mutant human ELA2 in transgenic mice will result in a SCN phenotype. A line of such mice would completely prove or disprove the first explanation as to why mutant murine ela2 mice do not show any signs of developing SCN. To test our hypothesis, we used a relatively new technology to engineer a site-specific G192ter mutation in this gene. The construct will later be injected into fertilized mice eggs to create a transgenic F1 mutant generation.

This new technology will make use of bacterial artificial chromosomes (BACs), which are stable and easy-to-use vectors for cloning large fragments of DNA. The BAC, RP11-646L4, containing the human neutrophil elastase gene and bordering CpG islands were obtained from the Human Genome Center. Our goal is to introduce the mutation into this BAC, clone and purify it, and inject it into fertilized eggs to see if it results in a mutant phenotype. Restriction digestion of the BAC using HindIII was used to determine the validity of the BAC. A PCR assay was also developed to specifically detect human, but not murine, ELA2. This assay was tested using RP11-646L4 as well as mouse genomic DNA to verify the human specificity of the primers. These primers will later be used on the F1 mutant generation to confirm the presence of the human ELA2 gene.

To create the G192ter mutation, we used a special line of DY380 recombineering bacteria that are able to homologously recombine ssDNA into the BACs. RP11-646L4 containing a chloramphenicol resistance gene was electroporated into the bacteria and grown on plates with the antibiotic. Colonies were selected and underwent several PCR assays and digestions to obtain and verify a viable line of transformed DY380 cells containing RP11-646L4. These cells will be used in another round of electroporation with a 73-mer ssDNA containing the desired G192ter mutation. To screen for recombinants containing the G192ter mutation, the cells will be first diluted and then pooled into 96 wells, each containing ~10 cells. Mutant primers with their 3’ end at the point of mutation that will only amplify the mutant BACs will be used to screen for these cells. The purified mutant BACs will then be injected into fertilized eggs, hopefully creating a comparable