224 CONSTRUCTION OF GENE TARGETING VECTORS FOR RAG-1 AND RAG-2

Gene targeting in mammalian cells has become a routine technique and is currently used to study gene function, create biomedical models, and generate potential tissue sources for xenotransplantation. Severe combined immunodeficiency (SCID) is a condition characterised by the absence of T cells and a lack of B cell function. Severe combined immunodeficiency affects ~1 out of every 100 000 infants. Autosomal recessive SCID can occur due to a mutation within the recombination activating genes (RAG-1/RAG-2) that play a role in recombination of immunoglobulins and T-cell receptors. Gene targeting has been used to create mouse models to study the effects of a RAG-1 or RAG-2 deficiency on the immune system. In 1992, Mombaerts et al. generated a homozygous mouse model of RAG-1 deficiency, whereas Shinkai et al. generated a homozygous mouse model of RAG-2. Both models resulted in the absence of mature T or B lymphocytes; which was concluded to be due to the lack of the ability to initiate the V(D)J recombination process. Because of the anatomical and physiological similarities between humans and pigs, a swine model of both RAG-1 and RAG-2 deficiency would have utility. We hypothesise that disruption of RAG-1, RAG-2, or both in swine will result in a SCID phenotype. A first step in the creation of a swine SCID model is to assemble targeting vectors. The objective of this work was to construct targeting vectors. To accomplish this goal, genomic DNA from porcine fetal fibroblasts was used to amplify a 6840-bp PCR product including the porcine RAG-1 gene. This fragment was cloned into TOPO pCR-XL (Invitrogen, Carlsbad, CA, USA). So that a mammalian G418 resistance cassette could be used for selection of targeting events, this plasmid was modified to remove the endogenous AphII gene (provides G418 resistance). The pKW4 contains LoxP (locus of X-ing over) sites that flank a G418 resistance cassette (based on mammalian codon usage), which is driven by a phosphoglycerate kinase (PGK) promoter (Lorson et al. 2011). This cassette was inserted into the RAG-1 gene to create the targeting construct pAB6. For RAG2, a 9466-bp PCR product i ncluding the RAG-2 gene was amplified and cloned into TOPO pCR-Blunt II (Invitrogen). The LoxP flanked G418 resistance cassette from pKW4 was inserted into the second exon of the RAG-2 gene sequence, creating the targeting construct pAB13. Further, diagnostic screening strategies were developed and validated to discriminate gene-targeting events from random integration. We report here 2 targeting vectors and validated screening methods for gene targeting in porcine fetal fibroblasts that have been validated for cloning. These vectors will be applied toward an effort to create a porcine SCID model. The implications of such a model include evaluation of basic immune function, evaluation of the innate immune system in vaccine efficacy, and organ transplantation.

Complicated colobomatous microphthalmia in the microphthalmic (mi/mi) mouse

A study of the development of the eye in the cinnamon mouse, homozygous for the gene for microphthalmia (mi), has shown that the microphthalmia is due to failure of secondary vitreous formation associated with a coloboma. The retina is dystrophic but there is a residual population of large ganglion cells and the optic nerve also contains ganglion cells. All these ganglion cells have cytoplasm similar to the retinal ganglion cells in the normal controls. It is postulated that they communicate with axons in the optic nerve. In addition, the outer epithelial layer of the eye cup, which normally becomes pigmented, forms retinal tissue in the homozygous mouse and this is also true of the dorsal part of the eyestalk near the eye.

Interaction between Kb and Q4 gene sequences generates the Kbm6 mutation.

Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr-to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules.

Interaction between Kb and Q4 gene sequences generates the Kbm6 mutation

Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr-to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules.

Studies of t6/t6 mouse embryos

The phenocritical period of the t6/t6 genome extends from the late blastocyst substages through the elongated egg-cylinder stage, whereas the lethal period begins at the short egg-cylinder-stage and extends through the elongated egg-cylinder stage. Most of the homozygous mouse embryos die during the short egg-cylinder stage. The viable egg-cylinder-staged t6/t6 embryos can be distinguished from their phenotypically wild-type litter-mates at both the light and electron microscopic levels. The distinguishing characteristics of these embryos are aberrantly arranged entodermal cells, excessive cytoplasmic lipid and crystal-containing mitochondria. These same features are also characteristic of those mutant embryos which are developmentally arrested at both the short and elongated egg-cylinder stages. Over 50% of the t6/t6 embryos can be identified as early as the late blastocyst substages by the presence of large, electron-dense cytoplasmic lipid droplets.

INHERITANCE OF ANTIBODY SPECIFICITY

Our data suggest that fine specificity of antihapten antibodies is a useful Mendelian marker of variable (V) genes. We found that some mouse strains (e.g., C57/BL6) consistently produced heteroclitic anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (relative affinity for related (4-hydroxy-5-iodo-3-nitrophenyl)acetyl and (4-hydroxy-3.5-dinitrophenyl)acetyl was always >2) while other strains (e.g., CBA) produced "conventional" anti-NP antibodies (relative affinities were consistently <1). 48 (CBA x C57BL/6)F1 mice were studied and most of them had heteroclitic anti-NP antibodies. They were backcrossed to the recessive CBA parent, and 87 backcross animals were similarly tested. Those heterozygous for the C57BL/6 heavy (H)-chain allotype were similar to the C57BL/6 and the F1 mice while mice homozygous for the CBA allotype were indistinguishable from the CBA. Such monogenic inheritance was observed only in the primary response. Predominance of allotype-linked genes in the control of the fine specificity characteristics was confirmed by immunizing selected homozygous mouse strains. These mice contained various mixtures of genes from C57BL, BALB/c, and other strains. Specificity of their anti-NP was exclusively determined by genes linked to the H-chain allotype, no influence could be attributed to other genes including the H-2-linked genes.