Limits...
Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) mice.

Stanford WL, Haque S, Alexander R, Liu X, Latour AM, Snodgrass HR, Koller BH, Flood PM - J. Exp. Med. (1997)

Bottom Line: To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A mice which are healthy and have normal numbers and percentages of hematopoietic lineages.However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates.This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7455, USA.

ABSTRACT
Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A-deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

Show MeSH

Related in: MedlinePlus

Gene-targeting of Ly-6A.2. (A) Strategy of Ly-6A gene targeting. A restriction map of the Ly-6A germ-line locus, the targeting  construct, and the targeted locus are depicted. The four exons of Ly-6A  are boxed and labeled. The Ly-6A targeting construct (pLy6ASDI-1) was  prepared by cloning the 4.5-kb EcoRI fragment encoding the Ly-6A.2  gene into the EcoRI site of pBluescript. The BclI fragment encoding exons 1–3 was excised and replaced with the pMC1neo gene. The HSV-tk  gene was cloned into the SalI site of the multiple cloning site of the targeting construct. Probes D, G, and H are PCR fragments using generated  PCR primers corresponding to sequences upstream or downstream from the  targeting construct, and probe B is the pMC1neo insert. The restriction  enzyme sites are designated as follows: BamHI (B), BclI (Bc), BglII (Bg),  EcoRI (R), EcoRV (V), HindIII (H), XbaI (X), and XmnI (Xmn). (B and  C) Detection of targeted and endogenous Ly-6A alleles by genomic  Southern blot analysis of EcoRV-digested DNA from a litter from a heterozygous cross using the 5′ probe D (B) and the 3′ probe H (C). The replacement of the 1.8-kb fragment encoding exons 1–3 with the NeoR reduces the size of the endogenous EcoRV band by 700 bp. The blot was  hybridized with probe H, stripped, and reprobed with probe D. The endogenous (5.2-kb) and targeted (4.5-kb) bands are marked with arrows.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199024&req=5

Figure 1: Gene-targeting of Ly-6A.2. (A) Strategy of Ly-6A gene targeting. A restriction map of the Ly-6A germ-line locus, the targeting construct, and the targeted locus are depicted. The four exons of Ly-6A are boxed and labeled. The Ly-6A targeting construct (pLy6ASDI-1) was prepared by cloning the 4.5-kb EcoRI fragment encoding the Ly-6A.2 gene into the EcoRI site of pBluescript. The BclI fragment encoding exons 1–3 was excised and replaced with the pMC1neo gene. The HSV-tk gene was cloned into the SalI site of the multiple cloning site of the targeting construct. Probes D, G, and H are PCR fragments using generated PCR primers corresponding to sequences upstream or downstream from the targeting construct, and probe B is the pMC1neo insert. The restriction enzyme sites are designated as follows: BamHI (B), BclI (Bc), BglII (Bg), EcoRI (R), EcoRV (V), HindIII (H), XbaI (X), and XmnI (Xmn). (B and C) Detection of targeted and endogenous Ly-6A alleles by genomic Southern blot analysis of EcoRV-digested DNA from a litter from a heterozygous cross using the 5′ probe D (B) and the 3′ probe H (C). The replacement of the 1.8-kb fragment encoding exons 1–3 with the NeoR reduces the size of the endogenous EcoRV band by 700 bp. The blot was hybridized with probe H, stripped, and reprobed with probe D. The endogenous (5.2-kb) and targeted (4.5-kb) bands are marked with arrows.

Mentions: The pl93+ plasmid containing a 4.5-kb EcoRI fragment encoding the Ly-6A.2 chromosomal gene has been described previously (27). The 1.7-kb fragment containing exons 1–3 was removed using methylation-sensitive BclI after cycling the plasmid through the dam−dcm− GM2163 Escherichia coli strain (E. coli; Genetic Stock Center, Department of Biology, Yale University, New Haven, CT). Oligonucleotide adapters (XSB-L1, 5′-TACACCCTCCCTTCATAGGAGCT-3′; XSB-U, 5′-CCTATGAAGGGAGGGTGTACTAG-3′) were used to anneal XhoI/SalI and BclI ends. XSB-L1 was kinased, annealed to XSB-U, and ligated to the BclI digested pl93+. To construct the pl93neo+ plasmid the 1.1-kb XhoI/SalI fragment of pMC1neo (Stratagene, La Jolla, CA), containing the neoR gene driven by the HSV-tk promoter and a polyoma enhancer, was inserted into the adapter-generated XhoI/SalI overhang of pl93+. In pl93neo+ the neoR transcriptional orientation is the same direction as Ly-6A. The HSV-tk cassette, obtained from Dr. M. Capecchi (Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT; reference 28), was inserted into the SalI site in the multiple cloning site of pl93neo+ to generate the targeting plasmid pLy6ASDI-1 (Fig. 1).


Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) mice.

Stanford WL, Haque S, Alexander R, Liu X, Latour AM, Snodgrass HR, Koller BH, Flood PM - J. Exp. Med. (1997)

Gene-targeting of Ly-6A.2. (A) Strategy of Ly-6A gene targeting. A restriction map of the Ly-6A germ-line locus, the targeting  construct, and the targeted locus are depicted. The four exons of Ly-6A  are boxed and labeled. The Ly-6A targeting construct (pLy6ASDI-1) was  prepared by cloning the 4.5-kb EcoRI fragment encoding the Ly-6A.2  gene into the EcoRI site of pBluescript. The BclI fragment encoding exons 1–3 was excised and replaced with the pMC1neo gene. The HSV-tk  gene was cloned into the SalI site of the multiple cloning site of the targeting construct. Probes D, G, and H are PCR fragments using generated  PCR primers corresponding to sequences upstream or downstream from the  targeting construct, and probe B is the pMC1neo insert. The restriction  enzyme sites are designated as follows: BamHI (B), BclI (Bc), BglII (Bg),  EcoRI (R), EcoRV (V), HindIII (H), XbaI (X), and XmnI (Xmn). (B and  C) Detection of targeted and endogenous Ly-6A alleles by genomic  Southern blot analysis of EcoRV-digested DNA from a litter from a heterozygous cross using the 5′ probe D (B) and the 3′ probe H (C). The replacement of the 1.8-kb fragment encoding exons 1–3 with the NeoR reduces the size of the endogenous EcoRV band by 700 bp. The blot was  hybridized with probe H, stripped, and reprobed with probe D. The endogenous (5.2-kb) and targeted (4.5-kb) bands are marked with arrows.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199024&req=5

Figure 1: Gene-targeting of Ly-6A.2. (A) Strategy of Ly-6A gene targeting. A restriction map of the Ly-6A germ-line locus, the targeting construct, and the targeted locus are depicted. The four exons of Ly-6A are boxed and labeled. The Ly-6A targeting construct (pLy6ASDI-1) was prepared by cloning the 4.5-kb EcoRI fragment encoding the Ly-6A.2 gene into the EcoRI site of pBluescript. The BclI fragment encoding exons 1–3 was excised and replaced with the pMC1neo gene. The HSV-tk gene was cloned into the SalI site of the multiple cloning site of the targeting construct. Probes D, G, and H are PCR fragments using generated PCR primers corresponding to sequences upstream or downstream from the targeting construct, and probe B is the pMC1neo insert. The restriction enzyme sites are designated as follows: BamHI (B), BclI (Bc), BglII (Bg), EcoRI (R), EcoRV (V), HindIII (H), XbaI (X), and XmnI (Xmn). (B and C) Detection of targeted and endogenous Ly-6A alleles by genomic Southern blot analysis of EcoRV-digested DNA from a litter from a heterozygous cross using the 5′ probe D (B) and the 3′ probe H (C). The replacement of the 1.8-kb fragment encoding exons 1–3 with the NeoR reduces the size of the endogenous EcoRV band by 700 bp. The blot was hybridized with probe H, stripped, and reprobed with probe D. The endogenous (5.2-kb) and targeted (4.5-kb) bands are marked with arrows.
Mentions: The pl93+ plasmid containing a 4.5-kb EcoRI fragment encoding the Ly-6A.2 chromosomal gene has been described previously (27). The 1.7-kb fragment containing exons 1–3 was removed using methylation-sensitive BclI after cycling the plasmid through the dam−dcm− GM2163 Escherichia coli strain (E. coli; Genetic Stock Center, Department of Biology, Yale University, New Haven, CT). Oligonucleotide adapters (XSB-L1, 5′-TACACCCTCCCTTCATAGGAGCT-3′; XSB-U, 5′-CCTATGAAGGGAGGGTGTACTAG-3′) were used to anneal XhoI/SalI and BclI ends. XSB-L1 was kinased, annealed to XSB-U, and ligated to the BclI digested pl93+. To construct the pl93neo+ plasmid the 1.1-kb XhoI/SalI fragment of pMC1neo (Stratagene, La Jolla, CA), containing the neoR gene driven by the HSV-tk promoter and a polyoma enhancer, was inserted into the adapter-generated XhoI/SalI overhang of pl93+. In pl93neo+ the neoR transcriptional orientation is the same direction as Ly-6A. The HSV-tk cassette, obtained from Dr. M. Capecchi (Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT; reference 28), was inserted into the SalI site in the multiple cloning site of pl93neo+ to generate the targeting plasmid pLy6ASDI-1 (Fig. 1).

Bottom Line: To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A mice which are healthy and have normal numbers and percentages of hematopoietic lineages.However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates.This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7455, USA.

ABSTRACT
Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A-deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A-deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

Show MeSH
Related in: MedlinePlus