168
Biochimica et Biophysica Acta, 1132 (1992) 168-176 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.1)(I
BBAEXP 92410
Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans Gwendolyn S. Adrian a, Damon C. Herbert a, LeAnn K. Robinson a, Christi A. Walter a, James M. Buchanan a, Erle K. Adrian a, Frank J. Weaker ~, Carlton A. Eddy b, Funmei Yang a and Barbara H. Bowman a " The Department of Cellular and Structural Biology and h The Department of Obstetrics and Gynecology, The UniL:ersity of Texas Health Science Center at San Antonio, San Antonio, TX (USA)
(Received 18 May 1992)
Key words: Transgenic mice; Total iron binding capacity; Transferrin; Albumin; Serum amyloid protein; C3; Aging; Acute phase reaction Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.
Introduction Analysis of human gene expression in the physiological background of aging can be carried out by production of transgenic mice that express human chimeric genes [1]. Expression of a human gene introduced into fertilized mouse eggs by microinjection can be followed during development, differentiation and aging. The in vivo response of injected genes to physiological regulators can be assessed in normal differentiated tissues that are frequently difficult to manipulate in vitro. The transgenic mouse model has been successfully used to study many complex mammalian mecha-
Correspondence to: G.S. Adrian, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7762, USA. Abbreviations: C3, complement component C3; CAT, chloramphenicol acetyl transferase; SAP, serum amyloid protein; TF, human transferrin; Tf, mouse transferrin; TIBC, total iron binding capacity.
nisms [2,3]. It provides the biochemical and physiological background of aging, expression in multiple cell types, and the necessary growth-regulating and early cell lineage factors. H u m a n gene expression can be followed in transgenic mice through maturity into aging, which in the mouse occurs in a relatively brief period of 18 to 28 months. Transferrin is a major plasma protein that transports ferric iron to target tissues throughout the body. The human transferrin (TF) gene demonstrates tissue specific expression and is mainly expressed in liver and brain, although extra-hepatic expression has been described in many tissue sites at lower levels (reviewed in Ref. 4). We chose the human transferrin gene to study in aging transgenic mice because of previous reports that suggested a decreased transferrin synthetic capacity in older people. Dybkjaer et al. [5] reported that transferrin levels in healthy human adults decrease with age. Transferrin and transferrin saturation in older people with iron deficiency anemia were found to be
169 significantly lower than in younger adults with iron deficiency anemia [6]. Total iron binding capacity (TIBC), which reflects transferrin concentration, was reported to decrease significantly during aging [7-9]. These studies raised the possibility of an age-related dysfunction in the expression of the human TF gene and a decreased protein synthetic capacity in older people which may underlie a reduced capacity to increase transferrin production in the iron-deficient state
[6]. The TF gene is developmentally controlled [10,11], regulated by heavy metals [12-14] and responds to hormonal [15-17] and inflammatory signals [18]; therefore, it has multiple circuits of regulation. Transferrin demonstrates conservation in function and structure [19-23], which makes it ideal to study during mammalian development and aging. While we [20,24-31] and others [14,21,32-35] have characterized transferrin gene structure and expression, relatively little is known about the DNA sequences involved in the regulation of TF gene expression during development and aging. Sequencing and correlating DNA sequences with gene expression have demonstrated that conserved sequences of DNA often located in the 5' flanking region of a gene serve to drive the expression of the gene at specific developmental stages and regulate its expression in specific tissues. Conserved DNA sequences in the human TF gene have been identified that respond in other genes to metals, to mitotic signals and to inflammatory factors [25]. In many well characterized genes, cis-regulatory sequences in the DNA have been shown to bind trans-acting nuclear proteins that can vary from tissue to tissue. Characteristic binding affinities of some of the cis-regulatory sequences have been studied in vitro in the human TF gene promoter [34]. The goal of this study was to use the transgenic mouse model to analyze specific DNA sequences of the regulatory region of human genes that respond to the aging process. A chimeric transferrin gene carrying approx. 670 base pairs of the 5' regulatory region of the human TF gene that had previously been introduced into the mouse genome was studied in the background of mammalian aging. The human TF DNA sequence directed reporter gene expression in aging transgenic mice that resembled the diminution of transferrin levels found in older humans. In both cases the products of liver TF gene expression decreased significantly during senescence. Examination of the TIBC (total iron binding capacity), which reflects transferrin concentration, in 701 humans between the ages of birth to 99 years confirmed previous reports [7] that the iron binding capacity in human plasma decreases with advanced age, and demonstrated that TIBC decreases significantly in humans 70 years old and continues to decrease through age 99. In comparison, the endogenous mouse Tf gene in the same transgenic
mice increased in expression during aging. The changes in expression of the human transgene and the mouse endogenous gene were not accompanied by a typical inflammatory response during aging. The results indicate that the human TF regulatory region directs expression similar to that found in the gene donor, and does not parallel the mouse Tf gene expression during aging. Thus, the transgenic model can be used to analyse age-related changes in mouse Tf and human TF chimeric genes simultaneously. Materials and Methods
Constructs of chimeric genes A chimeric gene, TF(0.67)CAT, consisting of 0.67 kb of the 5' regulatory region of the human TF gene fused to the bacterial reporter gene chloramphenicol acetyl transferase, was constructed previously [26,28]. Expression of the human TF(0.67)CAT gene in four independent founder lines of transgenic mice has been reported [26] and shown to occur mainly in liver and brain although, like the human and mouse endogenous genes, low expression is also found in other tissues. Expression of the TF(0.67)CAT transgene in one (A26X) of the four founder lines described before [26] is studied here during the aging process. The line was chosen because its tissue specific expression closely followed that seen in humans; the liver expression of the TF-CAT gene in A26X transgenic mice exceeded the expression in brain and all other tissues [26]. The TF-CAT gene in A26X was modulated by iron administration to transgenic mice. As in humans with hemochromatosis where transferrin decreases, TF-directed CAT activity in transgenic mice decreased after iron administration [26]. Microinjection of fertilized mouse eggs The methods used to develop transgenic mice have been published in detail by Hogan et al. [36]. Procedures used to insert the human TF-CAT chimeric genes into the mouse genome of the C57BL/6J line have previously been described [26]. Identification of transgenic mice At 4 weeks of age, 1 cm sections of tails from mice were examined to identify transgenic animals by polymerase chain amplification as described by Walter et al. [37]. Southern filter hybridization [38] was also used to determine copy number of genes integrated into the mouse genome. Maintenance of aging transgenic mice Transgenic mice were housed in a pathogen-free facility where a maximum number of individuals survive to their anticipated life span. Three different levels of disease protection were used. A non-patho-
170
[Non Pathogen-Free Microisolator Filter Tops (MET) Work In Laminar F)ow Workbench 1. 2.
(LFW)
Production of transgenic mice Identify transgenlc founder mice
INon P a t h o g e n - F r e e I (MFT, 1. 2. 3.
LFW)
M a t e f o u n d e r m i c e to g e n e r a t e Identify transgenic progeny Select lines for aging studies
a transgenic
line
1 !Intermediate Holding Area] (MFT, 1. 2. 3.
LFW)
Quarantine for 60 days (minimum) Mate transgeni¢ mice Determine pathogen status with sentinel
,Greatest Disease Protection 1. 2. 3. 4. 5. 6. 7.
MFT LFW Laminar flow racks Autoclaved cages, water, and food Very restricted access Personnel wear gowns, gloves Age mice
animals
l~le~an Breeding] t. 2.
MFT LFW
Fig. 1. Protocol for maintaining aging transgenic mice behind a pathogen-free barrier. MFT= microisolator filter tops; LFW= laminar flow workbench. gen-free room is used for housing mice purchased from commercial sources, including mice for superovulation and for surrogate motherhood. After identification, a transgenic founder mouse is transferred to another non-pathogen-flee room and bred to a nontransgenic mouse to establish that transgenic line. A transgenic F1 mouse, a nontransgenic mouse and their progeny are used to initiate pathogen-free animals. The breeding pair is transferred to an intermediate holding room for quarantine. Nontransgenic littermates of the F1 transgenic are also transferred to serve as sentinel animals. A quarantine is enforced for minimally 60 days. Every 30 days, sentinel animals are monitored for pathogens as described below. Pathogen-flee animals are transferred to a clean breeding room or a pathogen-flee aging room. As long as sentinel animals in the breeding area remain pathogenfree, animals born in the clean breeding room can be transferred to the aging room until the desired numbers of mice are available for ongoing studies. Mice are caged in the pathogen-flee aging area according to sex and projected date of sacrifice to reduce manipulation of animals. A summary of the steps toward maintaining mice in the pathogen-flee barrier is shown in Fig. 1. The mice are checked at the beginning of each day. The rooms are maintained at 23 + 2°C with 15 changes of flesh air per hour and are cleaned with sodium hypochlorite mixed with A33, a commercial disinfectant. Polycarbonate cages cleaned with antiseptic soap are filled with autoclaved bedding and are fitted with wire tops containing Purina lab chow and water twice a week. Except for sentinel cages, each cage in all rooms is equipped with a microisolator top and manipulations are performed with sterile tongs in a laminar flow
workbench. (Lab Products, Maywood, N J; Models 30909 and 30910). Nontransgenic mice are sacrificed monthly to monitor pathogens. These are tested for the presence of mouse hepatitis virus, Sendai virus, mycoplasma and internal/external parasites by veterinary staff in the D L A R (Department of Laboratory Animal Resources, The University of Texas Health Science Center at San Antonio). Additional tests are performed quarterly for gross pathology, histology and the presence of Salmonella by the DLAR, and every 6 months for 12 murine viruses by Microbiological Associates (Rockville, MD). Additional precautions are observed in pathogenfree areas housed in the D L A R facility. The rooms are located in a clean area which is under positive pressure relative to the corridors. This specific pathogen-free rodent housing area has limited access with a lock system. The lab chow has balanced nutrients after autoclaving, and has not been shown to contain toxic by-products. Cages are wrapped in groups of 4 and autoclaved as a unit. Water and water bottles are autoclaved. Additionally, the water is acidified to a pH of 2.5 to 2.8. Finally, gowns and gloves are worn by all personnel entering the area, access is limited to a very few people, and mice are housed in cages maintained in laminar flow cage racks (Forma Scientific, Marietta, OH; Model 1894). Using these techniques, we have attained between 81% and 94% survival of mice through 26 months of age behind the pathogen barrier.
CAT actiL,ity assays Quantitation of TF-directed CAT activity in tissue extracts from transgenic mice was determined by the procedure of Gorman et al. [39]. Assays of transgenic mice tissue carrying TF-CAT chimeric genes have been previously described [26]. Analysis of mouse plasma proteins during aging To determine levels of mouse transferrin (Tf), serum amyloid protein (SAP), complement factor 3 (C3) and albumin (Alb) in the sera of transgenic mice, rocket immunoelectrophoresis was carried out according to the procedure of Laurell [40]. Antibody preparations for mouse transferrin and albumin were obtained from Cappel-Organon Teknika (West Chester, PA). Antibody preparations for mouse SAP and rat C3 were obtained from Calbiochem Corporation (San Diego, CA) and United States Biochemical (Cleveland, OH), respectively. Samples were quantitated by measuring the peak height of the rocket immunoprecipitate of each sample. Isolation and blot hybridization of RNA from mouse tissues Tissue RNA was isolated by the acid guanidinium isothiocyanate-phenol-chloroform extraction procedure
171 [41] and identified by hybridization on dot blots [42] and Northern hybridization using a mouse TF cDNA probe or rat albumin cDNA. A Betagen Betascope 603 Blot analyzer was used for R N A quantitations.
Analysis of clinical laboratory data Determinations of TIBC in human plasma were from automated multi-test biochemical profiles provided to us by MetPath Laboratories, Teterboro, NJ. MetPath Laboratories routinely includes iron studies in its 27 component multitest analyses of blood samples. Comparison of 5' regions of human TF and mouse Tf genes D N A sequences in the noncoding 5' regions of the mouse T f [43] and the human T F [25] genes were compared by computer-assisted analysis using Pustell D N A / p r o t e i n sequence analysis by International Biotechnologies, New Haven, CT. Results
The A26X founder line of transgenic mice Southern analysis of D N A isolated from tails of the A26X founder line of transgenic mice revealed the line carried 5 copies of the 0.67 kb TF-CAT gene arranged in head to tail concatamers in the chromosomal site of integration. Expression of the human TF-CAT transgene in the tissues of 2-month-old A26X transgenic mice has been previously described [26]. As found in other founder lines carrying the TF(0.67)CAT construct, expression was mainly in liver and brain. The
4O
ira
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I-
0
30
Z I
Biochimica et Biophysica Acta, 1132 (1992) 168-176 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.1)(I
BBAEXP 92410
Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans Gwendolyn S. Adrian a, Damon C. Herbert a, LeAnn K. Robinson a, Christi A. Walter a, James M. Buchanan a, Erle K. Adrian a, Frank J. Weaker ~, Carlton A. Eddy b, Funmei Yang a and Barbara H. Bowman a " The Department of Cellular and Structural Biology and h The Department of Obstetrics and Gynecology, The UniL:ersity of Texas Health Science Center at San Antonio, San Antonio, TX (USA)
(Received 18 May 1992)
Key words: Transgenic mice; Total iron binding capacity; Transferrin; Albumin; Serum amyloid protein; C3; Aging; Acute phase reaction Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.
Introduction Analysis of human gene expression in the physiological background of aging can be carried out by production of transgenic mice that express human chimeric genes [1]. Expression of a human gene introduced into fertilized mouse eggs by microinjection can be followed during development, differentiation and aging. The in vivo response of injected genes to physiological regulators can be assessed in normal differentiated tissues that are frequently difficult to manipulate in vitro. The transgenic mouse model has been successfully used to study many complex mammalian mecha-
Correspondence to: G.S. Adrian, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7762, USA. Abbreviations: C3, complement component C3; CAT, chloramphenicol acetyl transferase; SAP, serum amyloid protein; TF, human transferrin; Tf, mouse transferrin; TIBC, total iron binding capacity.
nisms [2,3]. It provides the biochemical and physiological background of aging, expression in multiple cell types, and the necessary growth-regulating and early cell lineage factors. H u m a n gene expression can be followed in transgenic mice through maturity into aging, which in the mouse occurs in a relatively brief period of 18 to 28 months. Transferrin is a major plasma protein that transports ferric iron to target tissues throughout the body. The human transferrin (TF) gene demonstrates tissue specific expression and is mainly expressed in liver and brain, although extra-hepatic expression has been described in many tissue sites at lower levels (reviewed in Ref. 4). We chose the human transferrin gene to study in aging transgenic mice because of previous reports that suggested a decreased transferrin synthetic capacity in older people. Dybkjaer et al. [5] reported that transferrin levels in healthy human adults decrease with age. Transferrin and transferrin saturation in older people with iron deficiency anemia were found to be
169 significantly lower than in younger adults with iron deficiency anemia [6]. Total iron binding capacity (TIBC), which reflects transferrin concentration, was reported to decrease significantly during aging [7-9]. These studies raised the possibility of an age-related dysfunction in the expression of the human TF gene and a decreased protein synthetic capacity in older people which may underlie a reduced capacity to increase transferrin production in the iron-deficient state
[6]. The TF gene is developmentally controlled [10,11], regulated by heavy metals [12-14] and responds to hormonal [15-17] and inflammatory signals [18]; therefore, it has multiple circuits of regulation. Transferrin demonstrates conservation in function and structure [19-23], which makes it ideal to study during mammalian development and aging. While we [20,24-31] and others [14,21,32-35] have characterized transferrin gene structure and expression, relatively little is known about the DNA sequences involved in the regulation of TF gene expression during development and aging. Sequencing and correlating DNA sequences with gene expression have demonstrated that conserved sequences of DNA often located in the 5' flanking region of a gene serve to drive the expression of the gene at specific developmental stages and regulate its expression in specific tissues. Conserved DNA sequences in the human TF gene have been identified that respond in other genes to metals, to mitotic signals and to inflammatory factors [25]. In many well characterized genes, cis-regulatory sequences in the DNA have been shown to bind trans-acting nuclear proteins that can vary from tissue to tissue. Characteristic binding affinities of some of the cis-regulatory sequences have been studied in vitro in the human TF gene promoter [34]. The goal of this study was to use the transgenic mouse model to analyze specific DNA sequences of the regulatory region of human genes that respond to the aging process. A chimeric transferrin gene carrying approx. 670 base pairs of the 5' regulatory region of the human TF gene that had previously been introduced into the mouse genome was studied in the background of mammalian aging. The human TF DNA sequence directed reporter gene expression in aging transgenic mice that resembled the diminution of transferrin levels found in older humans. In both cases the products of liver TF gene expression decreased significantly during senescence. Examination of the TIBC (total iron binding capacity), which reflects transferrin concentration, in 701 humans between the ages of birth to 99 years confirmed previous reports [7] that the iron binding capacity in human plasma decreases with advanced age, and demonstrated that TIBC decreases significantly in humans 70 years old and continues to decrease through age 99. In comparison, the endogenous mouse Tf gene in the same transgenic
mice increased in expression during aging. The changes in expression of the human transgene and the mouse endogenous gene were not accompanied by a typical inflammatory response during aging. The results indicate that the human TF regulatory region directs expression similar to that found in the gene donor, and does not parallel the mouse Tf gene expression during aging. Thus, the transgenic model can be used to analyse age-related changes in mouse Tf and human TF chimeric genes simultaneously. Materials and Methods
Constructs of chimeric genes A chimeric gene, TF(0.67)CAT, consisting of 0.67 kb of the 5' regulatory region of the human TF gene fused to the bacterial reporter gene chloramphenicol acetyl transferase, was constructed previously [26,28]. Expression of the human TF(0.67)CAT gene in four independent founder lines of transgenic mice has been reported [26] and shown to occur mainly in liver and brain although, like the human and mouse endogenous genes, low expression is also found in other tissues. Expression of the TF(0.67)CAT transgene in one (A26X) of the four founder lines described before [26] is studied here during the aging process. The line was chosen because its tissue specific expression closely followed that seen in humans; the liver expression of the TF-CAT gene in A26X transgenic mice exceeded the expression in brain and all other tissues [26]. The TF-CAT gene in A26X was modulated by iron administration to transgenic mice. As in humans with hemochromatosis where transferrin decreases, TF-directed CAT activity in transgenic mice decreased after iron administration [26]. Microinjection of fertilized mouse eggs The methods used to develop transgenic mice have been published in detail by Hogan et al. [36]. Procedures used to insert the human TF-CAT chimeric genes into the mouse genome of the C57BL/6J line have previously been described [26]. Identification of transgenic mice At 4 weeks of age, 1 cm sections of tails from mice were examined to identify transgenic animals by polymerase chain amplification as described by Walter et al. [37]. Southern filter hybridization [38] was also used to determine copy number of genes integrated into the mouse genome. Maintenance of aging transgenic mice Transgenic mice were housed in a pathogen-free facility where a maximum number of individuals survive to their anticipated life span. Three different levels of disease protection were used. A non-patho-
170
[Non Pathogen-Free Microisolator Filter Tops (MET) Work In Laminar F)ow Workbench 1. 2.
(LFW)
Production of transgenic mice Identify transgenlc founder mice
INon P a t h o g e n - F r e e I (MFT, 1. 2. 3.
LFW)
M a t e f o u n d e r m i c e to g e n e r a t e Identify transgenic progeny Select lines for aging studies
a transgenic
line
1 !Intermediate Holding Area] (MFT, 1. 2. 3.
LFW)
Quarantine for 60 days (minimum) Mate transgeni¢ mice Determine pathogen status with sentinel
,Greatest Disease Protection 1. 2. 3. 4. 5. 6. 7.
MFT LFW Laminar flow racks Autoclaved cages, water, and food Very restricted access Personnel wear gowns, gloves Age mice
animals
l~le~an Breeding] t. 2.
MFT LFW
Fig. 1. Protocol for maintaining aging transgenic mice behind a pathogen-free barrier. MFT= microisolator filter tops; LFW= laminar flow workbench. gen-free room is used for housing mice purchased from commercial sources, including mice for superovulation and for surrogate motherhood. After identification, a transgenic founder mouse is transferred to another non-pathogen-flee room and bred to a nontransgenic mouse to establish that transgenic line. A transgenic F1 mouse, a nontransgenic mouse and their progeny are used to initiate pathogen-free animals. The breeding pair is transferred to an intermediate holding room for quarantine. Nontransgenic littermates of the F1 transgenic are also transferred to serve as sentinel animals. A quarantine is enforced for minimally 60 days. Every 30 days, sentinel animals are monitored for pathogens as described below. Pathogen-flee animals are transferred to a clean breeding room or a pathogen-flee aging room. As long as sentinel animals in the breeding area remain pathogenfree, animals born in the clean breeding room can be transferred to the aging room until the desired numbers of mice are available for ongoing studies. Mice are caged in the pathogen-flee aging area according to sex and projected date of sacrifice to reduce manipulation of animals. A summary of the steps toward maintaining mice in the pathogen-flee barrier is shown in Fig. 1. The mice are checked at the beginning of each day. The rooms are maintained at 23 + 2°C with 15 changes of flesh air per hour and are cleaned with sodium hypochlorite mixed with A33, a commercial disinfectant. Polycarbonate cages cleaned with antiseptic soap are filled with autoclaved bedding and are fitted with wire tops containing Purina lab chow and water twice a week. Except for sentinel cages, each cage in all rooms is equipped with a microisolator top and manipulations are performed with sterile tongs in a laminar flow
workbench. (Lab Products, Maywood, N J; Models 30909 and 30910). Nontransgenic mice are sacrificed monthly to monitor pathogens. These are tested for the presence of mouse hepatitis virus, Sendai virus, mycoplasma and internal/external parasites by veterinary staff in the D L A R (Department of Laboratory Animal Resources, The University of Texas Health Science Center at San Antonio). Additional tests are performed quarterly for gross pathology, histology and the presence of Salmonella by the DLAR, and every 6 months for 12 murine viruses by Microbiological Associates (Rockville, MD). Additional precautions are observed in pathogenfree areas housed in the D L A R facility. The rooms are located in a clean area which is under positive pressure relative to the corridors. This specific pathogen-free rodent housing area has limited access with a lock system. The lab chow has balanced nutrients after autoclaving, and has not been shown to contain toxic by-products. Cages are wrapped in groups of 4 and autoclaved as a unit. Water and water bottles are autoclaved. Additionally, the water is acidified to a pH of 2.5 to 2.8. Finally, gowns and gloves are worn by all personnel entering the area, access is limited to a very few people, and mice are housed in cages maintained in laminar flow cage racks (Forma Scientific, Marietta, OH; Model 1894). Using these techniques, we have attained between 81% and 94% survival of mice through 26 months of age behind the pathogen barrier.
CAT actiL,ity assays Quantitation of TF-directed CAT activity in tissue extracts from transgenic mice was determined by the procedure of Gorman et al. [39]. Assays of transgenic mice tissue carrying TF-CAT chimeric genes have been previously described [26]. Analysis of mouse plasma proteins during aging To determine levels of mouse transferrin (Tf), serum amyloid protein (SAP), complement factor 3 (C3) and albumin (Alb) in the sera of transgenic mice, rocket immunoelectrophoresis was carried out according to the procedure of Laurell [40]. Antibody preparations for mouse transferrin and albumin were obtained from Cappel-Organon Teknika (West Chester, PA). Antibody preparations for mouse SAP and rat C3 were obtained from Calbiochem Corporation (San Diego, CA) and United States Biochemical (Cleveland, OH), respectively. Samples were quantitated by measuring the peak height of the rocket immunoprecipitate of each sample. Isolation and blot hybridization of RNA from mouse tissues Tissue RNA was isolated by the acid guanidinium isothiocyanate-phenol-chloroform extraction procedure
171 [41] and identified by hybridization on dot blots [42] and Northern hybridization using a mouse TF cDNA probe or rat albumin cDNA. A Betagen Betascope 603 Blot analyzer was used for R N A quantitations.
Analysis of clinical laboratory data Determinations of TIBC in human plasma were from automated multi-test biochemical profiles provided to us by MetPath Laboratories, Teterboro, NJ. MetPath Laboratories routinely includes iron studies in its 27 component multitest analyses of blood samples. Comparison of 5' regions of human TF and mouse Tf genes D N A sequences in the noncoding 5' regions of the mouse T f [43] and the human T F [25] genes were compared by computer-assisted analysis using Pustell D N A / p r o t e i n sequence analysis by International Biotechnologies, New Haven, CT. Results
The A26X founder line of transgenic mice Southern analysis of D N A isolated from tails of the A26X founder line of transgenic mice revealed the line carried 5 copies of the 0.67 kb TF-CAT gene arranged in head to tail concatamers in the chromosomal site of integration. Expression of the human TF-CAT transgene in the tissues of 2-month-old A26X transgenic mice has been previously described [26]. As found in other founder lines carrying the TF(0.67)CAT construct, expression was mainly in liver and brain. The
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30
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