Production of transgenic rats by lentiviral transduction of male germ-line stem cells

Franklin K Hamra, Joel Gatlin, Karen M. Chapman, Dana M. Grellhesl, J. Victor Garcia, Robert E Hammer, David L. Garbers

Research output: Contribution to journalArticle

195 Citations (Scopus)

Abstract

Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin, The cells that bound to laminin represented ≈5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50% of pups in the F2 generation. Thus, by using these procedures, ≈30% of pups in the F1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

Original languageEnglish (US)
Pages (from-to)14931-14936
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number23
DOIs
StatePublished - Nov 12 2002

Fingerprint

Transgenic Rats
Germ Cells
Stem Cells
Transgenes
Testis
Population
Genetic Loci
Laminin
Vimentin
Spermatozoa
Collagen

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Production of transgenic rats by lentiviral transduction of male germ-line stem cells. / Hamra, Franklin K; Gatlin, Joel; Chapman, Karen M.; Grellhesl, Dana M.; Garcia, J. Victor; Hammer, Robert E; Garbers, David L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 23, 12.11.2002, p. 14931-14936.

Research output: Contribution to journalArticle

Hamra, Franklin K ; Gatlin, Joel ; Chapman, Karen M. ; Grellhesl, Dana M. ; Garcia, J. Victor ; Hammer, Robert E ; Garbers, David L. / Production of transgenic rats by lentiviral transduction of male germ-line stem cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2002 ; Vol. 99, No. 23. pp. 14931-14936.
@article{4054fd53da5248bcb8bfc9e0701b5875,
title = "Production of transgenic rats by lentiviral transduction of male germ-line stem cells",
abstract = "Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73{\%} of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin, The cells that bound to laminin represented ≈5{\%} of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50{\%} of pups in the F2 generation. Thus, by using these procedures, ≈30{\%} of pups in the F1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.",
author = "Hamra, {Franklin K} and Joel Gatlin and Chapman, {Karen M.} and Grellhesl, {Dana M.} and Garcia, {J. Victor} and Hammer, {Robert E} and Garbers, {David L.}",
year = "2002",
month = "11",
day = "12",
doi = "10.1073/pnas.222561399",
language = "English (US)",
volume = "99",
pages = "14931--14936",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "23",

}

TY - JOUR

T1 - Production of transgenic rats by lentiviral transduction of male germ-line stem cells

AU - Hamra, Franklin K

AU - Gatlin, Joel

AU - Chapman, Karen M.

AU - Grellhesl, Dana M.

AU - Garcia, J. Victor

AU - Hammer, Robert E

AU - Garbers, David L.

PY - 2002/11/12

Y1 - 2002/11/12

N2 - Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin, The cells that bound to laminin represented ≈5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50% of pups in the F2 generation. Thus, by using these procedures, ≈30% of pups in the F1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

AB - Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin, The cells that bound to laminin represented ≈5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50% of pups in the F2 generation. Thus, by using these procedures, ≈30% of pups in the F1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

UR - http://www.scopus.com/inward/record.url?scp=0037069345&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037069345&partnerID=8YFLogxK

U2 - 10.1073/pnas.222561399

DO - 10.1073/pnas.222561399

M3 - Article

C2 - 12391306

AN - SCOPUS:0037069345

VL - 99

SP - 14931

EP - 14936

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 23

ER -