# A Human cDNA Library for Functional Gene Replacement in Drosophila

> **NIH NIH R24** · BAYLOR COLLEGE OF MEDICINE · 2020 · $754,627

## Abstract

PROJECT SUMMARY
The aim of this proposal is to continue to develop a toolkit designed to facilitate the functional annotation of
human genes and disease associated variants through genetic studies in Drosophila melanogaster. We
initiated this project three years ago through support of an R24 funded by ORIP. The Drosophila genome
contains ~8,500 genes that are evolutionarily conserved in vertebrates including human. To model human
diseases, we typically start by creating a severe loss-of-function mutation of a fly gene that is likely to be an
ortholog of the human gene that is known or suspected to be pathogenic. We insert a SA-T2A-GAL4-polyA
artificial exon into an early intron common to all transcripts of the gene of interest (GOI) using CRISPR
mediated homologous recombination. This typically creates a strong loss-of-function allele that expresses the
GAL4 transactivator in the same spatial and temporal pattern as the mutated gene. Hence, a UAS-nuclear or
membrane GFP permits us to determine the cell types in which the gene is expressed through co-staining with
known cell identity markers or based on cellular morphology. Importantly, GAL4 often allows us to rescue the
phenotypes associated with the loss-of-function allele by driving a UAS-fly or human cDNA. If the human cDNA
rescues we can test human variants of interest for functionality in flies, an approach that has already greatly
helped in the identification of many new human diseases in the past few years. These experiments also allow
detailed functional analyses to better understand the pathogenic mechanisms and to test FDA approved or
experimental drugs. We have also produced a library of just over 2,000 T2A-GAL4 stocks and ~3,000 UAS-
human cDNAs lines to perform these experiments systematically. We assembled a library of 33,000 full length
human cDNAs from different sources, generated and sequenced ~4,000 plasmids containing the UAS-human
cDNA for transformation in the fly. Nearly 3,000 of these constructs have been inserted in the fly genome in
defined loci using the ΦC31 integrase, and transgenic stocks have been established. The UAS constructs are
available from the Drosophila Genomics Resource Center (DGRC) and the stocks are available from the
Bloomington and Kyoto stock centers. Here we propose to expand the UAS-human cDNA collection and clone
the remaining 4,000 human cDNAs of the 8,500 conserved genes and establish an additional 3,000 transgenic
stocks for distribution. We also propose to generate 1,000 SA-T2A-GAL4-polyA insertions in homologous fly
genes using a new method that we developed to accelerate the testing of the UAS-human cDNAs by the
research community and promote the systematic study of human disease associated genes. Our goal is to
provide molecular, genetic and transgenic resources to the fly research community and human geneticists to
accelerate the discovery of human diseases and help unravel human gene function.

## Key facts

- **NIH application ID:** 10047133
- **Project number:** 2R24OD022005-05
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** HUGO J BELLEN
- **Activity code:** R24 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $754,627
- **Award type:** 2
- **Project period:** 2016-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10047133

## Citation

> US National Institutes of Health, RePORTER application 10047133, A Human cDNA Library for Functional Gene Replacement in Drosophila (2R24OD022005-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10047133. Licensed CC0.

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